General cDNA Library Creation Protocol

Version Number: 4.1draft

> Production Dates: 7/27/06 to present
>
> Version Date: 7/27/06
>
> Author: Dean Ng
>
> Reviewed/Revised by: Chris Detter
>
> Erika Lindquist
>
> Paul Richardson
>
> Doug Smith
>
> Mei Wang

# Safety First

***Personal Protection Equipment (PPE) Requirements:***

***Safety glasses, lab coat, and nitrile gloves should be worn at all
times while performing this protocol. Additional safety equipment is
required at designated steps.***

**All procedures that uses phenol (a corrosive agent) *should be done in
a fume hood wearing tight fitting safety goggles. Waste containing
phenol should be disposed of in a separate designated container in the
fume hood. Phenol is a corrosive agent that burns skin upon contact, use
extreme care when handling phenol. ***

***Wear a face shield to minimize UV exposure to yourself. Turn off UV
immediately after each use.***

# Summary

The cDNA library construction process has been tested with a wide range
of samples. Since the inception of the cDNA program in 2004, we have
produced over 100 libraries from over 50 organisms. Complexity of
libraries produced range from 50K to \> 100 million. The process has
been modified to accommodate the wide range of samples that we get from
the community. We have incorporated controls at each crucial step to
monitor the progress and to help us improve process.

Our primary goal is to produce high quality sequence information of each
RNA that we receive. The process require multiple enzymatic steps that
are sensitive to impurities in the samples. We continue to look for ways
to further purify samples to ensure maximum complexity for all
libraries. In the mean time, quantity and quality of staring materials
are critical for eventual success for the final libraries.

PolyA RNA was reverse transcribed with superscript reverse transcriptase
using dT primer (5' GACTAGTTCTAGATCGCGAG CGGCCGCCCTTTTTTTTTTTTTTTVN-3').
cDNA was synthesized with E coli E coli DNA Ligase , E coli DNA
polymerase I , and E coli RNaseH. Ends were repaired with T4 DNA
polymerase.

SalI adapter (5'-TCGACCCACGCGTCCG-3' and 5'-P04-CGGACGCGTGGG-3') was
ligated to cDNA and the product was digested with NotI. Digested cDNA
were gel purified and directionally ligated into SalI and NotI digested
pCMVsport6 and transformed into ElectroMAX T1 DH10B cells. Sequence
template was prepared by rolling circle amplification (RCA)

This protocol is for the high-throughput production of cDNA libraries.
The starting material is purified polyA RNA and the finished product is
cDNA ligated into pCMVsport6 vector ElectroMAX T1 DH10B cells.

Procedure is modified from Invitrogen instructional manual for
"SuperScript plasmid system with gateway technology for cDNA synthesis
and Cloning" version B 29-september 2003#11108 and JGI 3 kb genomic
library creation protocols.

Sample data provided were collected during the production of library
CAAS.

Major changes from SOP3.0.0 include additional RNA purification steps to
remove polysaccharides and improved data entry forms.

# QC criteria

Starting material

[RNA-QC]{.underline}\
Define RNA quality and quantity standards to eliminate processing of
poor quality samples and to maximize library making success. 
Additionally early detection of problems in samples would speed up
feedback to the collaborator to resubmit samples.\
Samples are shipped as a salt precipitated in ethanol. If the sample is
in ethanol, sample is centrifuged at 4C Each sample is documented with
Bioanalyzer graph and gel and information\
2M LiCl extraction of RNA pellet to remove polysaccharides.\
PPT and/or resuspend entire contents of received tube, only remove an
aliquot of homogeneous sample.\
\
Bioanalyzer results:\
1. Quality -- Intact peaks of rRNA are good indicators of intact mRNA.
Most RNA will have only 28S and 18S RNA(figure 1A): however, many plants
have chloroplast rRNA that may show up as multiple rRNA
bands(figerue1B). In some cases, the 28S RNAis composed of two fragments
that comigrate with the 18S rRNA(figure 1C)

![](media/image1.pct){width="2.104861111111111in"
height="1.2916666666666667in"}![](media/image2.png){width="2.126388888888889in"
height="1.5319444444444446in"}![](media/image3.png){width="2.10625in"
height="1.4201388888888888in"}

Figure 1A. vertebrate rRNA bands. 1B. multiple rRNA bands in plants. 1C.
Single rRNA band in Lottia

2\. Improved quality of RNA after LiCl2 PPT

Not all RNA require LiCl2 ppt. However, large amounts of polysaccharides
that copurify with RNA often interferes with cDNA synthesis. LiCl2 PPT
of RNA samples improves the quality of the RNA (figures 2)

![](media/image4.pct){width="2.4784722222222224in"
height="1.2597222222222222in"}![](media/image5.pct){width="2.4819444444444443in"
height="1.2854166666666667in"}

Figure 2A. RNA prior to LiCl2 ppt. Figure 2B RNA after LiCl2 ppt

3\. Quantity - amount of polyA RNA isolated from sample in ug.

2-4ug polyA RNA with %rRNA \<20%(typical \<10%).

Ethanol precipitate DNA to concentrate samples to \>200 ng/ul, if
necessary.

Use NanoDrop or Agilent Bioanalyzer to verify RNA concentration.

> ![](media/image1.pct){width="2.30625in" height="1.4013888888888888in"}
>
> Figure 3. PolyA enriched RNA

Finished product:

cDNA sizes as determined by PCR should have sizes 0.5 to \>5 kb.

Colony count of ligation with insert \>10X of vector self ligation.

Insert rate \> 90%.

Complexity \> 100K

Insert sizes \>500bp.

Inserts containing rRNA and E coli seq should be \<10%

[\
cDNA Library PCR-QC SOP:]{.underline}\
Purpose: Define standards for quality assessment of PCR-QC gel for cDNA
libraries to limit time spent on poor quality libraries\
Store information in the cDNA tracking db; gel photo and numbers
outlined below.\
1. Minimum number of cfu screen = 20

2\. Minimum number of cfu with insert \>500bp = 11\
3. Maximum number of NULL PCR=7( to be confirmed)\
4. Maximum percentage of no insert clones per library = 20%\
5. Minimum complexity of 50K

# Materials and Reagents -- cDNA Synthesis 

+----------------------------+----------------------+-----------------+
| [Materials/Reage           | [Vendor]{.underline} | [Stock          |
| nts/Equipment]{.underline} |                      | Numb            |
|                            |                      | er]{.underline} |
+----------------------------+----------------------+-----------------+
| First Strand:              | Invitrogen           | 8248RT,         |
|                            |                      | 18248-013       |
| DEPC water                 | Invitrogen           |                 |
|                            |                      |                 |
| 5x first strand buffer     | Invitrogen           |                 |
| (1x=50mM Tris HCl          |                      |                 |
|                            | Operon               |                 |
| pH8.3, 75mM KCl, 5 mM      |                      |                 |
| MgCl~2~)                   | Invitrogen           |                 |
|                            |                      |                 |
| NotIdT15 (35uM)            | Invitrogen           |                 |
|                            |                      |                 |
| NotIdT15VN (35uM)          | Invitrogen           |                 |
|                            |                      |                 |
| 0.1M DTT                   |                      |                 |
|                            |                      |                 |
| 10mM dNTP                  |                      |                 |
|                            |                      |                 |
| superscript reverse        |                      |                 |
| transcriptase (200U/ul)    |                      |                 |
+----------------------------+----------------------+-----------------+
| Second strand:             | Invitrogen           | 8               |
|                            |                      | 248RT,18248-013 |
| 5x second strand buffer    | Invitrogen           |                 |
|                            |                      |                 |
| (1x=20mM Tris HCl pH6.9,   | Invitrogen           |                 |
| 90 mM KCl                  |                      |                 |
|                            | Invitrogen           |                 |
| 4.6 mM MgCl~2~, 0.15mM     |                      |                 |
| NAD+, 50mM                | Invitrogen           |                 |
| (NH~4~)~2~SO~4~.)          |                      |                 |
|                            | Invitrogen           |                 |
| 10mM dNTP                  |                      |                 |
|                            |                      |                 |
| E coli DNA Ligase 10 U/ul  |                      |                 |
|                            |                      |                 |
| E coli DNA polymerase I 10 |                      |                 |
| U/ul                       |                      |                 |
|                            |                      |                 |
| E coli RNaseH 2 U/ul       |                      |                 |
|                            |                      |                 |
| T4 DNA polymerase(5U/ul)   |                      |                 |
+----------------------------+----------------------+-----------------+
| SalI adapter ligation:     | Invitrogen           | 8               |
|                            |                      | 248RT,18248-013 |
| 5x T4 DNA ligase buffer    | Invitrogen /Operon   |                 |
| (1x=50mM Tris HCl pH7.6,   |                      |                 |
| 10mM MgCl~2~, 1mM ATP, 1mM |                      |                 |
| DTT, 5% PEG8000)           |                      |                 |
|                            |                      |                 |
| SalI adapter (1ug/ul,      |                      |                 |
| 110uM,                     |                      |                 |
|                            |                      |                 |
| 5'OH-TCGACCCACGCGTCCG::    |                      |                 |
|                            |                      |                 |
| 5'pCGGACGCGTGGG)           |                      |                 |
+----------------------------+----------------------+-----------------+
| NotI Digestion:            | Invitrogen           | 16              |
|                            |                      | 303-018(React3) |
| React 3 buffer 10x         | NEB                  |                 |
|                            |                      |                 |
| (1x = 50 mM Tris HCl (pH   |                      |                 |
| 8.0), 10 mM MgCl~2~ 100 mM |                      |                 |
| NaCl)                      |                      |                 |
|                            |                      |                 |
| NotI 15U/ul                |                      |                 |
+----------------------------+----------------------+-----------------+
| T4 ligase                  | Roche                | 10799009001     |
|                            |                      |                 |
|                            | Invitrogen           | 8               |
|                            |                      | 248RT,18248-013 |
+----------------------------+----------------------+-----------------+
| 10mM dNTP                  | MBI Fermentas        | R0192           |
+----------------------------+----------------------+-----------------+
| Phenol chloroform isoamyl  | Fluka                | 77617           |
| alcohol (PCI,25:24:1)      |                      |                 |
|                            | Sigma                | P4557           |
| Phenol in Tris             |                      |                 |
+----------------------------+----------------------+-----------------+
| Ultra Pure Ethidium        | Invitrogen           | 15585011        |
| Bromide (Carcinogen)       |                      |                 |
| (10mg/ml)                  |                      |                 |
+----------------------------+----------------------+-----------------+
| Marker 2 (lambda HindIII   | MBI Fermentas        | SM0101          |
| digested DNA 0.3ug/ul)     |                      |                 |
+----------------------------+----------------------+-----------------+
| Loading dye (30% glycerol, | JGI                  |                 |
| 0.08% bromophenol blue,    |                      |                 |
| 0.08% Xylene Cyanole FF)   |                      |                 |
+----------------------------+----------------------+-----------------+
| Pellet Paint®              | Novagene             | 69049           |
| Co-Precipitant             |                      |                 |
+----------------------------+----------------------+-----------------+
| Glycogen                   | Invitrogen           |                 |
+----------------------------+----------------------+-----------------+
| QIAquick Gel Extraction    | QIAGEN               | 28704,28706     |
| Kit (50,250)               |                      |                 |
+----------------------------+----------------------+-----------------+
| *[Equipment]{.underline}*  |                      |                 |
+----------------------------+----------------------+-----------------+
| SpeedVac                   |                      |                 |
+----------------------------+----------------------+-----------------+
| Agarose gel box and 1.5mm  | Owl                  | D-14            |
| 13 teeth combs             |                      |                 |
+----------------------------+----------------------+-----------------+
| NanoDrop spectrophotometer | NanoDrop             | ND1000          |
+----------------------------+----------------------+-----------------+

# Procedure -- cDNA Library construction 

All reagents/stock solutions should be prepared prior to the start of
the procedure. Once RNA is thawed to 4C, work quickly to prevent RNA
degradation. Wear gloves to protect samples from your hands and protect
your hand from harmful chemicals. Change gloves often. Put on a new pair
of gloves before handling critical samples. Change gloves after handling
tubes containing harmful agents such as phenol, Ethidium Bromide
(Carcinogen) or bacteria.

# polyA RNA purification

The protocol provides the essential info needed for an experienced user.

1.  Total RNA preparation

-   Centrifuge 200-300ug RNA 14000g 4C 30min if RNA is in ethanol

-   Wash pellect with 1 ml 70% ethanol, 14000g 4C 1min

-   Air dry pellet 10min 20C

-   Resuspend RNA in 200ul RNase free water

-   QC 1 ul of using Bioanalyzer RNA nano chip

-   Expect two rRNA bands and 500 to 2000 ng/ul

2.  LiCl2 ppt

-   Add 200ul 4M LiCl2 to 200ul RNA

-   Invert to mix

-   1h 4C

-   14000 rpm 4C 20 min

-   Wash pellet with 70%ethanol

-   14000rpm 4C 5min

-   Wash pellet with 70%ethanol

-   14000rpm 4C 5min

-   Air dry pellet 10min 20C

-   Resuspend RNA in 200ul RNase free water

-   QC 1 ul of using Bioanalyzer RNA nano chip

-   Expect two rRNA bands and 500 to 2000 ng/ul

3.  PolyA RNA isolation

-   Wash 100ul beads with 200ul hybrid mix 2x

-   Add 200ul RNA sample to 200ul of beads in hybrid mix.

-   20 min 20C with gentle mixing very 5min

-   Wash 4x 200ul wash mix

-   Elute with 12 ul elution mix

-   5min 20C

-   Remove sup to new tube

-   QC 1 ul of using Bioanalyzer RNA nano chip

-   Expect 50 to 200 ng/ul

# cDNA Synthesis Protocol

The protocol provides the essential info needed for an experienced user.
Refer to the specific sections for important details.

4.  Anneal polyA RNA to NotI primer

-   2 ug polyA RNA diluted in 8 ul nuclease free water

-   2ul 35uM NotI T15 or NotIT15VN primer-adapter

-   70C 10min, 4C 2 min

5.  First strand synthesis

-   4ul 5x FS buffer

-   2ul 0.1 M DTT

-   2 ul 10mM dNTP

-   37C 2 min

6.  Reverse transcription

-   2 ul (200u/ul) Superscript II Reverse transcriptase

-   37C 1h. 4C 5 min

7.  Second strand synthesis

-   93 ul DEPC water

-   30ul 5x SS buffer

-   3 ul 10mM dNTP

-   1ul E coli DNA ligase(10U/ul)

-   4ul E coli DNA polymerase(10U/ul)

-   1 ul E coli RnaseH (2 U/ul)

-   16C 2h. 4C 5 min

8.  End repair

-   2ul T4 DNA polymerase(5U/ul)

-   16C 5 min

-   10 ul 0.5M EDTA pH8

9.  Phenol chloroform isoamyl alcohol and EtOH PPT (Corrosive)

> PPE NOTE: Extraction should be done in hood wearing safety goggles.
> Waste should be disposed of in a separate designated container.
>
> Important Safety NOTE: Phenol is a corrosive agent which burns skin
> upon contact, practice extreme safety when performing the following
> ste**ps.**

-   160ul PCI, mix by pipetting gently and add to prespun phaselock
    heavy tubes.

-   Spin 5min 14000g RT.

-   Transfer top layer into tube (150uL).

-   1ul glycogen

-   75ul 7.5M NH~4~OAc

-   560uL EtOH, vortex, and spin 20min 14000g.

-   Wash pellet with 500ul 70% EtOH. Spin 14000 1 min

-   SpeedVac 10min RT.

10. SalI adapter ligation in tube "S" (50ul)

-   Add 25ul DEPC water to DNA pellet in tube "S."

-   10ul 5x T4 ligase buffer

-   10ul SalI adapter(110uM) Invitrogen or operon primers in 1x Reaciton
    3 buffer

-   5ul T4 DNA ligase(1U/ul)

-   16C 16h. Hold at 4C.

11. Phenol chloroform isoamyl alcohol and EtOH PPT (Corrosive)

-   50ul PCI, mix by pipetting gently and add to prespun phaselock heavy
    tubes.

-   Spin 5min RT. Transfer top layer into tube (50ul).

-   25ul 7.5M NH~4~OAc

-   188uL EtOH, vortex, spin 20min 14000g

-   Wash pellet with 500ul 70% EtOH. Spin 14000 1 min.

-   SpeedVac 10min RT

12. NotI digestion in tube "N"

-   41 ul DEPC water to DNA pellet in tube "N"

-   5ul 10x React 3 buffer

-   4ul NotI

-   37C 2h. 65C 20min. Hold at 4C.

13. EtOH PPT

-   25ul 7.5M NH~4~OAc

-   188uL ETOH, vortex, spin 20min 14000g

-   Wash pellet with 500ul 70% EtOH. Spin 14000 1 min and remove
    remaining sup

-   SpeedVac 10min RT.

14. Gel 1.1% agarose in 1XTAE with 0.5 ug/ml Ethidium Bromide
    (Carcinogen).

-   Add 10ul water to DNA pellet.

-   10ul 1x Sample buffer

-   Load 20ul on gel.

-   cut gel slices A, B

15. Gel extraction using Qiagen spin column

-   Process gel slices A and B

-   Elute each with 30ul EB (50C).

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-   Determine DNA concentration by Bioanalyzer (1-15ng/ul).

-   Save cDNA at --20C

16. Ligation

-   1uL cut vector (pCMVsport6 -sap-SalI,50ng)

-   9.8ul (\~100ng) insert

-   6.2 ul premix with Roche ligase (1.6ul 10x, 2.4 ul 30%PEG,1.2 ul
    ligase)

-   4C 5 min, 16C 16h, Hold 4C.

17. Phenol extraction (Corrosive)

-   Add 85ul TE0.1

-   Mix with 100ul Tris saturated phenol

-   Transfer to prespun phase Lock light tubes

-   Spin 1 min 14000rpm RT

-   Add sup to tubes containing 1ul pellet paint, 10ul NaOAc, 100ul EtOH

-   -80C 1h

-   Spin 14000rpm 30min

-   Wash pellet with 1 ml 70% EtOH, spin 14000rpm 1 min RT

-   Speed vac 5 min

-   Respuspend in 15 ul TE 0.1

-   Vortex RT 10min 800rpm

18. Transformation

-   Electroporate 1ul with 20ul T1 E. coli DH10b cells. 2000V

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-   Recover with 980ul SOC.

-   37C 1h, rotator speed 5

-   4C 5min

-   Add 140ul 80% Glycerol. Gently mixed

-   Plate 10ul. Barcode tf stock tubes and store at --80C.

-   Incubate late 37C 16h.

19. Library QC

-   Count cfu. 100 cfu/ul glycerol stock 10K/gly stock 1.5M/ligation 
    30M/ug.

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-   PCR 22 Cfu /ligation. Include positive and negative controls.

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-   Gel 120V 30min. Pass library if insert rate \>85%. Insert size \>
    0.5kb

# DATA 

Fill out the following data sheet for permanent record.

Date: \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Absolutely RNA purification kit Lot#
\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_Kit#\_\_\_\_\_\_\_\_\_\_\_\_\_

1.  RNA sample descriptions

+--------+--------+-----+-----+---+--------+-------+-------+-------+
| S      | RNA    | \   | ul  | u | \      | ul    | ug    | Com   |
| pecies | date   | [RN |     | g | [RNA\] |       |       | ments |
|        |        | A\] |     |   | ng/ul  |       |       |       |
|        |        | ng  |     | ( |        |       |       |       |
|        |        | /ul |     | e |        |       |       |       |
|        |        |     |     | x |        |       |       |       |
|        |        |     |     | p |        |       |       |       |
|        |        |     |     | e |        |       |       |       |
|        |        |     |     | c |        |       |       |       |
|        |        |     |     | t |        |       |       |       |
|        |        |     |     | e |        |       |       |       |
|        |        |     |     | d |        |       |       |       |
|        |        |     |     | ) |        |       |       |       |
+--------+--------+-----+-----+---+--------+-------+-------+-------+
| FHM    | 1/     | 1   | 200 | 2 | 129    | 12    | 1.5   | none  |
|        | 1/2006 | 000 |     | 0 |        |       |       |       |
|        |        |     |     | 0 |        |       |       |       |
+--------+--------+-----+-----+---+--------+-------+-------+-------+
|        |        |     |     |   |        |       |       |       |
+--------+--------+-----+-----+---+--------+-------+-------+-------+
|        |        |     |     |   |        |       |       |       |
+--------+--------+-----+-----+---+--------+-------+-------+-------+
|        |        |     |     |   |        |       |       |       |
+--------+--------+-----+-----+---+--------+-------+-------+-------+
|        |        |     |     |   |        |       |       |       |
+--------+--------+-----+-----+---+--------+-------+-------+-------+
|        |        |     |     |   |        |       |       |       |
+--------+--------+-----+-----+---+--------+-------+-------+-------+
|        |        |     |     |   |        |       |       |       |
+--------+--------+-----+-----+---+--------+-------+-------+-------+
|        |        |     |     |   |        |       |       |       |
+--------+--------+-----+-----+---+--------+-------+-------+-------+
|        |        |     |     |   |        |       |       |       |
+--------+--------+-----+-----+---+--------+-------+-------+-------+
|        |        |     |     |   |        |       |       |       |
+--------+--------+-----+-----+---+--------+-------+-------+-------+
|        |        |     |     |   |        |       |       |       |
+--------+--------+-----+-----+---+--------+-------+-------+-------+
|        |        |     |     |   |        |       |       |       |
+--------+--------+-----+-----+---+--------+-------+-------+-------+
|        |        |     |     |   |        |       |       |       |
+--------+--------+-----+-----+---+--------+-------+-------+-------+
|        |        |     |     |   |        |       |       |       |
+--------+--------+-----+-----+---+--------+-------+-------+-------+
|        |        |     |     |   |        |       |       |       |
+--------+--------+-----+-----+---+--------+-------+-------+-------+
|        |        |     |     |   |        |       |       |       |
+--------+--------+-----+-----+---+--------+-------+-------+-------+
|        |        |     |     |   |        |       |       |       |
+--------+--------+-----+-----+---+--------+-------+-------+-------+
|        |        |     |     |   |        |       |       |       |
+--------+--------+-----+-----+---+--------+-------+-------+-------+
|        |        |     |     |   |        |       |       |       |
+--------+--------+-----+-----+---+--------+-------+-------+-------+
|        |        |     |     |   |        |       |       |       |
+--------+--------+-----+-----+---+--------+-------+-------+-------+
|        |        |     |     |   |        |       |       |       |
+--------+--------+-----+-----+---+--------+-------+-------+-------+
|        |        |     |     |   |        |       |       |       |
+--------+--------+-----+-----+---+--------+-------+-------+-------+
|        |        |     |     |   |        |       |       |       |
+--------+--------+-----+-----+---+--------+-------+-------+-------+
|        |        |     |     |   |        |       |       |       |
+--------+--------+-----+-----+---+--------+-------+-------+-------+
|        |        |     |     |   |        |       |       |       |
+--------+--------+-----+-----+---+--------+-------+-------+-------+
|        |        |     |     |   |        |       |       |       |
+--------+--------+-----+-----+---+--------+-------+-------+-------+
|        |        |     |     |   |        |       |       |       |
+--------+--------+-----+-----+---+--------+-------+-------+-------+
|        |        |     |     |   |        |       |       |       |
+--------+--------+-----+-----+---+--------+-------+-------+-------+
|        |        |     |     |   |        |       |       |       |
+--------+--------+-----+-----+---+--------+-------+-------+-------+
|        |        |     |     |   |        |       |       |       |
+--------+--------+-----+-----+---+--------+-------+-------+-------+
|        |        |     |     |   |        |       |       |       |
+--------+--------+-----+-----+---+--------+-------+-------+-------+
|        |        |     |     |   |        |       |       |       |
+--------+--------+-----+-----+---+--------+-------+-------+-------+
|        |        |     |     |   |        |       |       |       |
+--------+--------+-----+-----+---+--------+-------+-------+-------+
|        |        |     |     |   |        |       |       |       |
+--------+--------+-----+-----+---+--------+-------+-------+-------+
|        |        |     |     |   |        |       |       |       |
+--------+--------+-----+-----+---+--------+-------+-------+-------+
|        |        |     |     |   |        |       |       |       |
+--------+--------+-----+-----+---+--------+-------+-------+-------+
|        |        |     |     |   |        |       |       |       |
+--------+--------+-----+-----+---+--------+-------+-------+-------+
|        |        |     |     |   |        |       |       |       |
+--------+--------+-----+-----+---+--------+-------+-------+-------+
|        |        |     |     |   |        |       |       |       |
+--------+--------+-----+-----+---+--------+-------+-------+-------+

Bioanalyzer pdf file name
\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Bioanalyzer pdf file name
\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

file location
\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

2.  Library information

Library kit: Lot \# \_\_\_\_\_\_\_\_\_\_\_\_\_Kit# \_\_\_\_\_\_\_\_\_\_
manufacturer : Invitrogen

> NotI Lot \# \_\_\_\_\_\_\_\_\_\_\_\_\_Kit# \_\_\_\_\_\_\_\_\_\_
> manufacturer : NEB/invitrogen
>
> Dt primer Lot \# \_\_\_\_\_\_\_\_\_\_\_\_\_Kit# \_\_\_\_\_\_\_\_\_\_
> manufacturer : Operon/Invtrogen
>
> DtVN primer Lot \# \_\_\_\_\_\_\_\_\_\_\_\_\_Kit# \_\_\_\_\_\_\_\_\_\_
> manufacturer : Operon
>
> SalI adapter Lot \# \_\_\_\_\_\_\_\_\_\_\_\_\_Kit#
> \_\_\_\_\_\_\_\_\_\_ manufacturer : Operon/Invtrogen
>
> pCMVsport6 Lot \# \_\_\_\_\_\_\_\_\_\_\_\_\_Kit# \_\_\_\_\_\_\_\_\_\_
> manufacturer : Invitrogen

Date: \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ comp Cell DH10B T1
Resist lot# \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

+------+-------+------+-----------+-----------+-----+-------+---+---+
| Lib  | Lig   | In   | Tf        | Vol       | Cfu | Cfu   | f | n |
| rary | ation | sert | bardcode  | ligation  |     | QC    | a | u |
| name |       | size |           | used      | /1  |       | i | l |
|      |       | (kb) |           |           | 0ul | (\>15 | l | l |
|      |       |      |           | (uL)/     |     | and   |   |   |
|      |       |      |           | total vol | (   | \     |   |   |
|      |       |      |           |           | \>1 | >80%) |   |   |
|      |       |      |           |           | 00) |       |   |   |
+------+-------+------+-----------+-----------+-----+-------+---+---+
| CAAS | 101B  | 2    | P0####    | 1 /15     | 400 | 18    | 1 | 3 |
+------+-------+------+-----------+-----------+-----+-------+---+---+
| P    |       |      |           |           |     |       |   |   |
| uc19 |       |      |           |           |     |       |   |   |
| 10pg |       |      |           |           |     |       |   |   |
+------+-------+------+-----------+-----------+-----+-------+---+---+
| No   |       |      |           |           |     |       |   |   |
| in   |       |      |           |           |     |       |   |   |
| sert |       |      |           |           |     |       |   |   |
+------+-------+------+-----------+-----------+-----+-------+---+---+
| Con  |       |      |           |           |     |       |   |   |
| trol |       |      |           |           |     |       |   |   |
| in   |       |      |           |           |     |       |   |   |
| sert |       |      |           |           |     |       |   |   |
+------+-------+------+-----------+-----------+-----+-------+---+---+
|      |       |      |           |           |     |       |   |   |
+------+-------+------+-----------+-----------+-----+-------+---+---+
|      |       |      |           |           |     |       |   |   |
+------+-------+------+-----------+-----------+-----+-------+---+---+
|      |       |      |           |           |     |       |   |   |
+------+-------+------+-----------+-----------+-----+-------+---+---+
|      |       |      |           |           |     |       |   |   |
+------+-------+------+-----------+-----------+-----+-------+---+---+
|      |       |      |           |           |     |       |   |   |
+------+-------+------+-----------+-----------+-----+-------+---+---+
|      |       |      |           |           |     |       |   |   |
+------+-------+------+-----------+-----------+-----+-------+---+---+
|      |       |      |           |           |     |       |   |   |
+------+-------+------+-----------+-----------+-----+-------+---+---+
|      |       |      |           |           |     |       |   |   |
+------+-------+------+-----------+-----------+-----+-------+---+---+
|      |       |      |           |           |     |       |   |   |
+------+-------+------+-----------+-----------+-----+-------+---+---+
|      |       |      |           |           |     |       |   |   |
+------+-------+------+-----------+-----------+-----+-------+---+---+
|      |       |      |           |           |     |       |   |   |
+------+-------+------+-----------+-----------+-----+-------+---+---+
|      |       |      |           |           |     |       |   |   |
+------+-------+------+-----------+-----------+-----+-------+---+---+
|      |       |      |           |           |     |       |   |   |
+------+-------+------+-----------+-----------+-----+-------+---+---+
|      |       |      |           |           |     |       |   |   |
+------+-------+------+-----------+-----------+-----+-------+---+---+
|      |       |      |           |           |     |       |   |   |
+------+-------+------+-----------+-----------+-----+-------+---+---+
|      |       |      |           |           |     |       |   |   |
+------+-------+------+-----------+-----------+-----+-------+---+---+
|      |       |      |           |           |     |       |   |   |
+------+-------+------+-----------+-----------+-----+-------+---+---+
|      |       |      |           |           |     |       |   |   |
+------+-------+------+-----------+-----------+-----+-------+---+---+
|      |       |      |           |           |     |       |   |   |
+------+-------+------+-----------+-----------+-----+-------+---+---+
|      |       |      |           |           |     |       |   |   |
+------+-------+------+-----------+-----------+-----+-------+---+---+
|      |       |      |           |           |     |       |   |   |
+------+-------+------+-----------+-----------+-----+-------+---+---+
|      |       |      |           |           |     |       |   |   |
+------+-------+------+-----------+-----------+-----+-------+---+---+
|      |       |      |           |           |     |       |   |   |
+------+-------+------+-----------+-----------+-----+-------+---+---+
|      |       |      |           |           |     |       |   |   |
+------+-------+------+-----------+-----------+-----+-------+---+---+
|      |       |      |           |           |     |       |   |   |
+------+-------+------+-----------+-----------+-----+-------+---+---+
|      |       |      |           |           |     |       |   |   |
+------+-------+------+-----------+-----------+-----+-------+---+---+
|      |       |      |           |           |     |       |   |   |
+------+-------+------+-----------+-----------+-----+-------+---+---+

> QC gel pictures name
> \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_
>
> File location
> \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

3.  cfu pcr

Tag polymerase Lot \# \_\_\_\_\_\_\_\_\_\_\_\_\_Kit#
\_\_\_\_\_\_\_\_\_\_ manufacturer : Roche

> dNTP Lot \# \_\_\_\_\_\_\_\_\_\_\_\_\_Kit# \_\_\_\_\_\_\_\_\_\_
> manufacturer :
>
> primer1 Lot \# \_\_\_\_\_\_\_\_\_\_\_\_\_Kit# \_\_\_\_\_\_\_\_\_\_
> manufacturer : Operon
>
> primer2 Lot \# \_\_\_\_\_\_\_\_\_\_\_\_\_Kit# \_\_\_\_\_\_\_\_\_\_
> manufacturer : Operon
>
> neg control Lot \# \_\_\_\_\_\_\_\_\_\_\_\_\_Kit# \_\_\_\_\_\_\_\_\_\_
> manufacturer : JGI
>
> pos control Lot \# \_\_\_\_\_\_\_\_\_\_\_\_Date# \_\_\_\_\_\_\_\_\_\_
> manufacturer : JGI
>
> gel file name
> \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_
>
> lane legend

  --------- ------- ------- ------ ------ ------- ------- -------- ----------- -------
  library   Pass?   \>500   fail   null   total   Blank   No       Lib         1.5kb
                    bp                                    insert               

  CAAA      pass    17      1      3      21      A1      A2       A3-A23      A24

                                                  A1      A2       A3-A23      A24

                                                  B1      B2       B3-B23      B24

                                                  C1      C2       C3-C23      C24

                                                  D1      D2       D3-D23      D24

                                                  E1      E2       E3-E23      E24

                                                  F1      F2       F3-F23      F24

                                                  G1      G2       G3-G23      G24

                                                  H1      H2       H3-H23      H24

                                                                               

                                                                               

                                                                               

                                                                               

                                                                               

                                                                               
  --------- ------- ------- ------ ------ ------- ------- -------- ----------- -------

> QC gel pictures name
> \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_
>
> File location \_\_Octopus/cloning technology/dean/pictures/2006
>
> \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

4.  1 plate sequence qc 384 wells

+------+-------+-----+-----------+-----------+-----------+----+----+
| Lib  | V80 % | Q20 | #clusters | Largest   | Largest   | P  | n  |
| rary |       | bp  |           | cluster   | cluster   | as | ee |
| name | \<10  |     | \>250     |           |           | s/ | d# |
|      |       | \>  |           |           |           |    | pl |
|      |       | 600 |           |           |           | fa | at |
|      |       |     |           |           |           | il | es |
+------+-------+-----+-----------+-----------+-----------+----+----+
| CAAS | 1     | 650 | 295       | actin     | 25        | pa | 36 |
|      |       |     |           |           |           | ss |    |
+------+-------+-----+-----------+-----------+-----------+----+----+
|      |       |     |           |           |           |    |    |
+------+-------+-----+-----------+-----------+-----------+----+----+
|      |       |     |           |           |           |    |    |
+------+-------+-----+-----------+-----------+-----------+----+----+
|      |       |     |           |           |           |    |    |
+------+-------+-----+-----------+-----------+-----------+----+----+
|      |       |     |           |           |           |    |    |
+------+-------+-----+-----------+-----------+-----------+----+----+
|      |       |     |           |           |           |    |    |
+------+-------+-----+-----------+-----------+-----------+----+----+
|      |       |     |           |           |           |    |    |
+------+-------+-----+-----------+-----------+-----------+----+----+
|      |       |     |           |           |           |    |    |
+------+-------+-----+-----------+-----------+-----------+----+----+
|      |       |     |           |           |           |    |    |
+------+-------+-----+-----------+-----------+-----------+----+----+
|      |       |     |           |           |           |    |    |
+------+-------+-----+-----------+-----------+-----------+----+----+
|      |       |     |           |           |           |    |    |
+------+-------+-----+-----------+-----------+-----------+----+----+
|      |       |     |           |           |           |    |    |
+------+-------+-----+-----------+-----------+-----------+----+----+
|      |       |     |           |           |           |    |    |
+------+-------+-----+-----------+-----------+-----------+----+----+
|      |       |     |           |           |           |    |    |
+------+-------+-----+-----------+-----------+-----------+----+----+
|      |       |     |           |           |           |    |    |
+------+-------+-----+-----------+-----------+-----------+----+----+
|      |       |     |           |           |           |    |    |
+------+-------+-----+-----------+-----------+-----------+----+----+
|      |       |     |           |           |           |    |    |
+------+-------+-----+-----------+-----------+-----------+----+----+
|      |       |     |           |           |           |    |    |
+------+-------+-----+-----------+-----------+-----------+----+----+
|      |       |     |           |           |           |    |    |
+------+-------+-----+-----------+-----------+-----------+----+----+
|      |       |     |           |           |           |    |    |
+------+-------+-----+-----------+-----------+-----------+----+----+
|      |       |     |           |           |           |    |    |
+------+-------+-----+-----------+-----------+-----------+----+----+
|      |       |     |           |           |           |    |    |
+------+-------+-----+-----------+-----------+-----------+----+----+
|      |       |     |           |           |           |    |    |
+------+-------+-----+-----------+-----------+-----------+----+----+
|      |       |     |           |           |           |    |    |
+------+-------+-----+-----------+-----------+-----------+----+----+
|      |       |     |           |           |           |    |    |
+------+-------+-----+-----------+-----------+-----------+----+----+
|      |       |     |           |           |           |    |    |
+------+-------+-----+-----------+-----------+-----------+----+----+
|      |       |     |           |           |           |    |    |
+------+-------+-----+-----------+-----------+-----------+----+----+
|      |       |     |           |           |           |    |    |
+------+-------+-----+-----------+-----------+-----------+----+----+
|      |       |     |           |           |           |    |    |
+------+-------+-----+-----------+-----------+-----------+----+----+

# Procedure -- Phenol chloroform isoamyl alcohol (PCI) extraction and ethanol precipitation

**[Phenol Extraction (Corrosive: Handle with care)]{.underline}**

***PPE NOTE: Extraction should be done in hood wearing safety goggles.
Waste should be disposed of in a separate designated container.***

***Important Safety NOTE: Phenol is a corrosive agent which burns skin
upon contact, practice extreme safety when performing the following
steps.***

1.  Note the sample volume.

2.  Spin phaselock tube 10000 rpm 2 min.

3.  Add equal volume PCI at 4C to sample (store at 4C in an approved
    refrigerator).

4.  Pipette up and down gently to mix.

5.  Spin 14000rpm 5 min at room temperature (RT).

6.  Transfer top layer to a pre labeled tube. Discard gel and PCI in
    proper waste container.

7.  Add 1 ul glycogen regardless of vol or amount of DNA or RNA in tube.

8.  Add ½ vol NH~4~OAc (4C).

9.  Add 2.5 vol 100% ethanol.

10. Vortex gently to mix.

11. Spin 20 min 14000 rpm with hinge side out to facilitate pellet
    identification after spin,

12. Look for a white \~1 mm size pellet on the hinge side. This pellet
    should be present even if there were no DNA/RNA. If not present,
    check to see if all reagents were added. Spin again if needed to see
    the pellet.

13. Remove tube from centrifuge gently and remove sup right away with a
    mircropipette tip pointing away from the pellet. Check to make sure
    the pellet remains after you remove the supernatant.

14. Add 500ul 70% ethanol RT.

15. Spin 1 min 14000 rpm RT.

16. Transfer sup with a micropipette. Make sure the pellet remains.

17. Spin 30 sec the pellet again to collect residual liquid that may
    have been on the side wall.

18. Remove remaining sup with a fine tip pipette such as one for p20.

19. Speedvac 10 min with 5 min low heat.

20. Resuspend translucent pellet with water as required by pipetting up
    and down 20 times. Make sure the pellet is fully dissolved.

  ------------------ ----------------- ----------------- -----------------
  Sample vol         50ul              100ul             150ul

  NH~4~OAc           25ul              50ul              75ul

  Glycogen           1ul               1ul               1ul

  Ethanol            188ul             375ul             562ul
  ------------------ ----------------- ----------------- -----------------

# Procedure -- Agarose Gel Separation 

# (Modified from General 3kb Library Creation Protocol 6.1)

1.  Make one \~100 ml 1.1% TAE agarose gel with 0.5 ug/ml Ethidium
    Bromide (Carcinogen).

2.  (use 12 cm x 14 cm gel tray and 1.5mm 13 tooth combs) for every 5
    DNA samples. (Load a sample every other well)

3.  Resuspend pellet with 10ul nuclease free water and add 10µl of 1X
    loading dye (in 30% glycerol) to each tube.

4.  Load 15 µl of cDNA product into each well. Save 5ul for future
    analysis (tube G).

5.  Load 15µl of lambda HindIII size marker (marker 2 only) into the
    left and right wells.

6.  Run for 180 min at 20V.

-   *It is very important to run the gel at 1.5V/cm to maximize
    separation of small molecular weight adapters away from the cDNA.
    After this happens you can image and cut out the desired 0.5 -2 and
    2-4 kb bands for each sample.*

7.  Image gel for no longer than 1-3 sec. to minimize UV exposure and
    save photos of both pre-cut and post-cut gels. cDNA are normally not
    visible under UV. The adapters should run below 500bp. Make sure not
    to include the adapter into your gel pieces.

8.  Using the UV transluminator, cut the cDNA lanes with scalpel blade
    0.7kb, 2kb, and 8kb.Cut lane into \~200mg pieces and put each slice
    into its pre-labeled 1.5-ml microfuge tube. Pre-weigh an empty
    microfuge tube. Weight the gel in tube to estimate gel volume.

-   *Important! Minimize UV exposure to yourself (use long sleeves lab
    coats and face shield) and to the samples (use laminated paper each
    section of samples not being cut). Turn off UV after cutting each
    band. Transfer agar pieces to tubes with normal light. Review cut
    gel to make sure the right pieces of gel were transferred.*

  ----------------------- -----------------------------------------------
  A                       2-8

  B                       0.7-2
  ----------------------- -----------------------------------------------

9.  Gel purify fragments A and B according to Qiagen protocol.

![](media/image8.png){width="2.2444444444444445in"
height="1.3833333333333333in"}

Figure 4. cDNA size fractionation. Uncut gel (left) and gel after gel
excision(right).

# Procedure -- Gel Purification 

# (Modified from General 3kb Library Creation Protocol 6.1)

1.  Heat QG buffer in \~60^o^C oven \~10 min before using. You can do
    this while you are cutting out each slice. This will just help with
    the melting of the gel slices. Add 600µl of warm Buffer QG to gel
    weighing less than 200mg. Make sure gel and QG buffer are at the
    bottom of tube.

2.  Incubate in shaker heating block at 40^o^C for 10-15 minutes (invert
    every 2 min). Dissolved gel solution must remain yellow to have
    proper pH. If Pink, adjust pH with 10 µl of 3M NaOAc, pH5.

3.  Following the Qiagen protocol steps, add the \~750µl sample to the
    Qiagen spin columns. Spin at \~13000 rpm for 1 min. remove filtrate
    from bottom waste tube. If sample is more than 750 µl, reload once,
    spin, and remove filtrate.

4.  Add 750 µl of wash PE wash buffer to each spin column. Let sit for 1
    min. Spin for 1 minute at 13,000rpm. remove filtrate from bottom
    waste tube.

5.  Spin for 1 minute at 13,000rpm to dry spin column.

6.  To elute DNA, put spin columns in new 1.5-ml tubes and add 30µl of
    warm (\~40-50 ^o^C) EB (elution buffer).

7.  Let sit 5 min. then spin or 1 minute at 13,000rpm.

8.  QC 2µl of each sample with NanoDrop. A260/280 should be close to
    1.8.\[DNA\] should be 5-20ng/ul. PPT if needed to increase \[DNA\]
    to \>5ng/ul.

# Procedure -- Vector Ligation 

# (Modified from General 3kb Library Creation Protocol 6.1)

1.  Default vector is pCMVsport6 precut with SalI and NotI

2.  Alternate vector is pMCL200cDNA vector (\~50 ng) was cut with NotI,
    dephosphorylated with SAP, and then digested with SalI. Single
    dephosphorylation is needed because vector insert has SalI adapter
    that is not phosphorylated.

3.  In a 1.5-ml centrifuge tube on ice, prepare a cocktail mix of:

    -   Vortex well and quick spin tube before dispensing.

4.  Add 9.8 µl of the gel purified cDNA sample (\~100 ng) into a
    designated well of a 96 well PCR plate. Mix the DNA with the vector
    by mixing up and down gently a few times with the pipette.

5.  Include one well with 9.8ul water as no insert negative control.

6.  Include one well with 1 ul 1.7 kb control insert + 8.8ul water as
    positive control.

7.  Dispense 5.2 µl of cocktail mix to each sample in the PCR plate (on
    the side wall of each well). Mix by gently pipetting up and down 10x
    Seal plate. Quick spin the plate 1000g 30sec.

8.  In a PE 9700 PCR machine set up the following ligation protocol
    times:

    -   *4C 5min,16^o^C for 16h, 70^o^C for 15 minutes, and a hold of
        4^o^C*. Reaction can stay overnight at 4 ^o^C if needed, but
        should be stored at -20 ^o^C for long-term storage.

# Materials and Reagents -- Transformation

  -------------------------------------------- ------------------------ ---------------------
  [Materials/Reagents/Equipment]{.underline}   [Vendor]{.underline}     [Stock
                                                                        Number]{.underline}

  *[Disposables]{.underline}*                                           

  Gene Pulse Cuvette 0.1 cm electrode gap      BioRad                   165-2089

  Falcon 14 ml Polypropylene Tube              Becton Dickinson         352059

  Cryogenic Vial                               Corning                  430289

  LB Amp 100 X-gal Plates                      Teknova                  L4902

                                                                        

  *[Reagents]{.underline}*                                              

  ElectroMAX™ DH10B™ T1 Phage-Resistant        Invitrogen               12033-015
  Competent Cells (box of 5 x 100 µl ea.)                               

  SOC Medium                                   Teknova                  0166-10

                                                                        

  *[Equipment]{.underline}*                                             

  Gene Pulser II                               BioRad                   \-

  Pulse Controller Plus                        BioRad                   \-
  -------------------------------------------- ------------------------ ---------------------

# Procedure -- Electro Transformation 

# (Modified General 3kb Library Creation Protocol 6.1) 

1\. [Equipment Settings (BioRad Pulse Controller):]{.underline}

-   Low range: 200

-   High range: ∞ (not used)

-   Capacitance: 25

-   Voltage: 2.0 kV

2\. [Transformation]{.underline}

1.  Place cuvette on ice \>2 min.

2.  Thaw T1 ElectroMax DH10B competent cells on ice (each tube contains
    100µl, enough for 5 reactions).

3.  Suspend 1µl of ligation product as a drop on the side wall of a
    chilled cuvette placed sideways on ice.

4.  Thaw cells completely on ice.

5.  Stir competent cells with pipette tip a few times.

-   Do not pipette up and down to mix the cells.

    1.  Slowly pipette 20µl competent cells to the ligation mix on the
        sidewall. Slowly Pipette up and down once to mix cells with
        ligation mix. Avoid creating bubbles with pipetting as they may
        cause the cuvette to arc during electroporation.

    2.  Tap cuvette on bench to allow the cells to settle to the bottom.
        (check to see that cells distribute evenly at the bottom of the
        cuvette and that there are no bubble).

    3.  Electroporate at 2.00 kV. Typical time reading is between 3.4
        and 4.3 msec. No cuvette will give \>5msec reading. Arced
        cuvette will give \<2 msec reading.

```{=html}
<!-- -->
```
-   Arced tubes may have reduced efficiency. If it is due to bubbles,
    redo electroporation. If arced 2^nd^ time, it may be a result of
    high salt in mix. Try ½ uL or PPT in NH4OAc and ethanol to remove
    salt and try again.

    1.  Add 980µl of SOC to cells in cuvette. Pipette gently once to
        mix.

```{=html}
<!-- -->
```
-   *Important! Transfer electroporation within 10 seconds.*

    1.  Transfer SOC mixture with p1000 into 15ml (17X100) culture
        tubes.

    2.  Incubate within rotating wheel at 37 ^o^C for 1 hour. Speed code
        set to 5.

    3.  After incubation, place on ice (no more than one hour) until
        ready to plate on agar plates.

3\. [Plating]{.underline}

1.  When the 60-minute transformation incubation begins, warm one
    LB/Amp/IPTG/X-gal agar plate per library at 37^o^C in an incubator
    to dry upside down and open.

2.  Add 140 µl 80% glycerol to 1000ul transformation in SOC at 4C to
    make a \~ 1140 10% glycerol transformation stock for each library.

3.  Mix gently and transfer into prebarcoded tubes.

4.  Plate 10µl of transformation glycerol mixture onto the appropriately
    labeled bioassay:

    a.  Add 10 to 20 beads onto pre-warmed plates.

    b.  Mix 10µl of transformation to 1000µl SOC in a microfuge tube

    c.  Pipette mixture onto the plate with 20beads.

    d.  Rock plate to spread evenly across the entire plate.

5.  Record number on data sheet.

6.  Store transformation glycerol mixture immediately at -80 ^o^C.

7.  Incubate the plates upside down in 37^o^C incubator for \~18 hrs.

8.  Count colonies and determine the complexity of ligation reaction
    (total \# of colonies in ligation).

+----------------------+-------------+--------------+-----------------+
| Sample name          | Time        | Cfu/10ul     | Tf eff          |
|                      | constant    |              |                 |
|                      |             |              | Cfu/ug          |
+----------------------+-------------+--------------+-----------------+
| Puc19 (10pg)         | 3.5         | 400          | 4.6e9           |
+----------------------+-------------+--------------+-----------------+
| No insert            | 4.2         | 17           | N/a             |
+----------------------+-------------+--------------+-----------------+
| 101B (example)       | 4.3         | 400          | 14e6            |
+----------------------+-------------+--------------+-----------------+

# Materials and Reagents -- PCR QC

  -------------------------------------------- ------------------------ ---------------------
  [Materials/Reagents/Equipment]{.underline}   [Vendor]{.underline}     [Stock
                                                                        Number]{.underline}

  *[Disposables]{.underline}*                                           

  96-well PCR plate                            USA Scientific           1402-9708

  *[Reagents]{.underline}*                                              

  Taq DNA Polymerase (10KU)                    Amersham Biosciences     

  10X PCR Buffer                               Amersham Biosciences     

  10mM dNTP mix                                MBI Fermentas            27079963

  DnM13-F primer (GTAAAACGACGGCCAGT)           Operon                   Custom

  DnM13-R primer (AGGAAACAGCTATGACCAT)         Operon                   custom

  *[Equipment]{.underline}*                                             

  GeneAmp PCR System 9700                      Perkin Elmer (Applied    \-
                                               Biosystems)              
  -------------------------------------------- ------------------------ ---------------------

# Procedure -- PCR for QC of Library Insert Size

# (Modified General 3kb Library Creation Protocol 6.1) 

1.  Set up a PE 9700 with the following colony PCR program (This is a 4
    hr 35 min protocol):

> 94 ^o^C -- 4 min.
>
> 94 ^o^C -- 30 sec.
>
> 55 ^o^C -- 30 sec. 35 cycles
>
> 68 ^o^C -- \*4 min.
>
> (\*modify with + 5 sec per cycle for to final extension time of 6:55.)
>
> 4 ^o^C -- Hold

2.  Make up the following PCR master mix for each sample (x120 for a
    96-well plate): Keep on ice.

+-----------------------+---------------+---------------+-------------+
|                       | 1X            | 120X          | 440X        |
+-----------------------+---------------+---------------+-------------+
| H~2~O                 | 16.85 µl      | 2022 µl       | 7414 µl     |
+-----------------------+---------------+---------------+-------------+
| ##                    | 2.00 µl       | 240 µl        | 880 µl      |
| ###### 10X PCR Buffer |               |               |             |
+-----------------------+---------------+---------------+-------------+
| 10mM dNTP (MBI)       | 0.40 µl       | 48 µl         | 176 µl      |
+-----------------------+---------------+---------------+-------------+
| F primer (10 pmol/µl) | 0.28 µl       | 34 µl         | 123 µl      |
+-----------------------+---------------+---------------+-------------+
| R primer (10 pmol/µl) | 0.28 µl       | 34 µl         | 123 µl      |
+-----------------------+---------------+---------------+-------------+
| Taq (Amersham)        | 0.28 µl       | 34 µl         | 123 µl      |
+-----------------------+---------------+---------------+-------------+
| Total Volume:         | 20.09 µl      | 2412 µl       | 8839 µl     |
+-----------------------+---------------+---------------+-------------+

3.  Dispense 20µl of the PCR mix into each well needed of the PCR plate.
    Keep plate on ice.

4.  Spin down the plate.

5.  Make a visual check to make sure all of the wells have mix in them.

6.  Using pipette tips only, pick the desired number of colonies per
    library (usually 22) into their own well. Include + and -- pcr
    controls.

7.  Quick spin plate and place in PCR machine with above protocol.

8.  start PCR

9.  Pour 1.1 % TAE gel.

10. After the 4:35 hrs run, add 10ul 1x loading dye to each well.

11. Mix before loading with up/down motion using pipette.

12. Load 10µl.

13. ![](media/image10.png){width="3.9153543307086616in"
    height="1.2255905511811023in"}Run for \~ 30 min at 120V.

Figure 5. PCR of cfu from each lib to determine insert sizes.

# References

+-----------------------+-----------------------+-----------------------+
| Source                | Authors               | DATE                  |
+-----------------------+-----------------------+-----------------------+
| General cDNA Library  | Dean Ng               | 3/12/05               |
| Creation Protocol     |                       |                       |
| 1.02                  |                       |                       |
+-----------------------+-----------------------+-----------------------+
| General 3kb Library   | Chris Detter,         | 4/19/04               |
| Creation Protocol 6.1 |                       |                       |
|                       | Eileen Dalin,         |                       |
|                       |                       |                       |
|                       | Jamie Jett,           |                       |
|                       |                       |                       |
|                       | Doug Smith            |                       |
+-----------------------+-----------------------+-----------------------+
| pCMVsport6            | ######## Invitrogen   | 9/29/2003             |
|                       |                       |                       |
| Superscript Plasmid   |                       |                       |
| System with Gateway   |                       |                       |
| Technology for cDNA   |                       |                       |
| Synthesis and cloning |                       |                       |
|                       |                       |                       |
| Version B 11108       |                       |                       |
+-----------------------+-----------------------+-----------------------+
| Reagent kit Guide     | ########              | 04/2004               |
|                       |  Agilent Technologies |                       |
| RNA 65000 Nano Assay  |                       |                       |
+-----------------------+-----------------------+-----------------------+
| Handbook for gel      | ######## Qiagen       |                       |
| extraction            |                       |                       |
+-----------------------+-----------------------+-----------------------+

# Oligonucleotides

\>NotI T15

GACTAGTTCTAGATCGCGAG CGGCCGCCCTTTTTTTTTTTTTTT

\>NotI T15VN

GACTAGTTCTAGATCGCGAG CGGCCGCCCTTTTTTTTTTTTTTTVN

\>M13-F primer (pcr and seq)

GTAAAACGACGGCCAGT

\>M13-R primer (pcr and seq)

AGGAAACAGCTATGACCAT

\>cmvsport6FW491

AACTCTCAAGCAGCAAGCA

\>cmvsport6REV1075

GACGCAAATGGGCGGTAG

\>cmvsport6FW398

ATGAGCCTTGGGACTGTGAA

\>cmvsport6REV1230

ATGGTGATGCGGTTTTGG

Oligos for future development:

\>NotI N6 future development

GACTAGTTCTAGATCGCGAG CGGCCGCCCNNNNNN

\>NotI-adapter future development

GACTAGTTCTAGATCGCGAG

\>SalI-primer future development

GAGAGAACAAGTCGACCCACGCGTCCG

\>SalI-adapter-top future development

TCGACCCACGCGTCCG

\>salI-adapter-bot future development

CGGACGCGTGGG

Sample lot numbers

  ------------------- -------------- ------------- ---------- ------------------------------
  reagents            manufacturer   cat           lot        description

  Absolutely RNA      Stratagene     400806        360759     polyA RNA purification kit

  superscriptII       Invitrogen     18248-013     1336098    Reverse transcriptase

  cDNA kit            Invitrogen     18248-013     1336098    

  cDNA kit            Clontech       634903        6080007    

  SalI adapter        Operon         custom        none       SalI adapter (110uM,\
                                                              5'OH-TCGACCCACGCGTCCG::\
                                                              5'pCGGACGCGTGGG)

  NotI                NEB            R0189M        50         

  T4 Ligase           Roche          10799009001   11790325   
                                                   30 june    
                                                   2007       

  NotIdtvN            operon         custom        none       5' GACTAGTTCTAGATCGCGAG
                                                              CGGCCGCCCTTTTTTTTTTTTTTTVN-3

  electromaxDH10BT1   invtrogen      12033-015     1317264    
  ------------------- -------------- ------------- ---------- ------------------------------

# SOP Approval

  ----------------------- ----------------------- -----------------------
  DEPARTMENT              APPROVED BY             DATE

  Lab Supervisor                                  

  Research and                                    
  Development                                     

  Instrumentation                                 

  QC                                              

  Purchasing                                      

  EH and S                                        

  Dept Head of Prod Seq                           
  ----------------------- ----------------------- -----------------------