16S/18S rRNA PCR Library Creation

> Version Number: 1.1
>
> Production Start Date: 4/24/07
>
> Version 1.1 Date: 03/17/08
>
> Author(s): Tanja Woyke, Doug Smith
>
> Reviewed/Revised by: Julianna Chow, Karla Ikeda, Eileen Dalin

# Summary

This protocol is to (i) check the purity of DNA used for genome shotgun
sequencing of single organisms and (ii) evaluate the diversity of an
environmental DNA sample.

# EH&S

JGI employees performing this procedure must wear a lab coat, safety
glasses, and gloves at all times.

# Materials & Reagents -- PCR amplification and Cloning

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

  *[Disposables]{.underline}*                                                 

  384-well PCR Microplate, Blue                    Abgene                     TF-0384/B

  Clear Adhesive Plate Sealers                     Edge BioSystems            48461

                                                                              

  *[Reagents]{.underline}*                                                    

  Milli-Q Water                                    Millipore Milli-Q System   \-

  27F primer (AGAGTTTGATCCTGGCTCAG)                IDT or Operon or your      \-
                                                   favorite                   

  4aF primer (TCCGGTTGATCCTGCCRG)                  IDT or Operon or your      \-
                                                   favorite                   

  1391R primer (GACGGGCRGTGWGTRCA)                 IDT or Operon or your      \-
                                                   favorite                   

  515F (GTGCCAAGCAGCCGCGGTAA)                      IDT or Operon or your      \-
                                                   favorite                   

  1209R (GGGCATCACAGACCTG)                         IDT or Operon or your      \-
                                                   favorite                   

  *Escherichia coli* *K-12* genomic DNA            ATCC                       10798D-5

  *Archaeoglobus fulgidis* genomic DNA             ATCC                       49558D

  ***Saccharomyces cerevisiae genomic DNA***       Isolated In house          []{.mark}

  Easy-A High-Fidelity PCR Master Mix              Stratagene                 600640

  6X loading dye                                   See reagent/stock          \-
                                                   preparation                

  Agarose, for routine use                         Sigma                      A9539-250G

  50X TAE Buffer                                   Invitrogen                 24710-030

  Ultra Pure Ethidium Bromide (10mg/ml)            Invitrogen                 15585011

  1 Kb DNA ladder                                  Invitrogen                 15615-016

  TOPO TA Cloning Kit for Sequencing               Invitrogen                 K4580-40

                                                                              

  *[Equipment]{.underline}*                                                   

  CLP Agarose gel box (12cm x 14cm) and two 25     CLP                        75.1214-MT-25D
  teeth combs                                                                 
  ------------------------------------------------ -------------------------- -----------------------

# Procedure -- PCR amplification and Cloning

> ***NOTE:** All reagents/stock solutions should be prepared prior to
> the start of the procedure.*
>
> ***NOTE:** Cloning should be performed on the same day as PCR
> amplification.*

1.  **[16S & 18S rRNA Gene PCR Amplification]{.underline}**

    1.  Add **\~100 ng** of DNA template to the bottom of a clean,
        well-labeled 96-well plate and per primer set required. Less DNA
        can be used and still produces amplified product.

> ***NOTE:** There are three primer sets for rRNA gene amplification as
> follows:*
>
> *27F/1391R for bacterial 16S rRNA gene amplification (BAC 16S)*
>
> *4aF/1391R for archaeal 16S rRNA gene amplification (ARCH 16S)*
>
> *515F/1209R for eukaryotic 18S rRNA gene amplification (EUK 18S)*
>
> ***NOTE:** draw out which samples require amplification with which
> primer set and use different plates or plate sections for each primer
> set required.*
>
> ***NOTE:** For environmental samples: leave 2 blank rows per row
> used!*

2.  Add a positive control and a negative control per primer set used:

Add **\~10 ng** of control DNA to the bottom of the 96-well plate:

> *E. coli* DNA for BAC 16S, *A. fulgidis* genomic DNA for ARCH 16S,
> ***Saccharomyces cerevisiae*** genomic DNA for EUK 18S

3.  Bring each sample volume up to **18.9 µl** with nuclease free H~2~O.

4.  Set-up the following PCR master mix for each primer combination for
    the amount of samples plus positive and negative controls. Keep
    plate on ice.

  ------------------------------------ ---------------------- -----------------------
                                       **[1X]{.underline}**   **[12X]{.underline}**

  F primer (10 µM)                     1.8 µl                 21.6 µl

  R primer (10 µM)                     1.8 µl                 21.6 µl

  Easy-A Master Mix                    22.5 µl                270 µl

  Total Volume:                        **26.1 µl**            **313.2 µl**
  ------------------------------------ ---------------------- -----------------------

5.  Dispense **26.1 µl** of the PCR mix into each PCR plate well with
    template DNA. Keep plate on ice.

6.  Spin down the plate.

7.  Make a visual check to make sure all of the wells have mix in them
    (**45 µl**/ well).

8.  Using the multi-channel pipettor, mix the samples by pipetting up
    and down gently. Be careful of your well location and of
    contamination.

9.  **[For environmental samples only:]{.underline}** using the
    multi-channel pipettor transfer 2 times 15 µl/ sample into the rows
    below.

> ***NOTE:** You will end up with **3x 15 µl**/ environmental sample.*

10. Quick spin plate.

11. Set up a PE 9700 with the following PCR program (This is a 1.5 h
    protocol):

> 94 ^o^C -- 3 min.
>
> **94 ^o^C -- 30 sec.**
>
> **55 ^o^C -- 30 sec. 20 cycles**
>
> **68 ^o^C -- 90 sec.**
>
> 72^o^C -- 10 min.
>
> 4 ^o^C -- ∞

12. **[For environmental samples only:]{.underline}** after the PCR
    reaction, pool the samples back into one **45 µl** aliquot/ sample
    and mix the samples by pipetting up and down gently. Be careful of
    your well location and of contamination.

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2.  **[Amplification QC using an Agarose Gel]{.underline}**

    1.  Transfer a **10 µl** aliquot per sample to a fresh PCR plate (or
        tubes).

    2.  Add **5 µl** of 1X loading dye per sample.

    3.  Vortex and spin quickly to collect the sample.

    4.  Load **15 µl** of sample onto 1% agarose gel containing Ethidium
        Bromide and save the remaining sample for cloning (on ice). Load
        **10 µl** of 1 Kb DNA ladder between the samples.

> ***NOTE:** use smallest comb size for the agarose gel (i.e. 25 teeth
> combs for 12cm x 14cm agarose gel box).*
>
> ![](media/image1.png){width="0.375in"
> height="0.3020833333333333in"}***CAUTION!** Use proper safety
> precautions while handling Ethidium Bromide. This is a
> carcinogen/irritant.*

5.  Run gel for \~ 40 min at 120V.

6.  Image gel and check for proper inserts at **\~1.4 kb** (BAC16S),
    **\~1.4 kb** and/or **\~2.1 kb** (ARCH16S) and **\~0.7 kb** (EUK
    18S). Make sure there is no non-specific amplification.

> ***NOTE:** you should see a single discrete band (exceptions are
> possible; i.e. archaeal 16S rRNA genes with introns; some variations
> in rRNA gene size).*
>
> *If you see more than a discrete band of the expected size or smearing
> from your PCR, you either (i) optimize your PCR to eliminate
> non-specific amplification or (ii) gel-purify your fragment prior to
> cloning (avoid nuclease contamination when gel purifying!)*
>
> ***NOTE:** make sure your positive control is positive and your
> negative controlis negative.*

7.  Discard the gel in the appropriately labeled waste.

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3.  **[Ligation of Amplified Product]{.underline}**

    1.  Add **1-4 µl** of fresh PCR product to the bottom of a clean,
        well-labeled 96-well plate (do not include negative controls).

> *NOTE: use PCR product volumes based on the intensity of the PCR
> product on the gel (i.e. use 4 µl, if very faint).*

2.  Add **1 µl** of dilute salt solution. (1:4 dilution of stock salt
    solution)

3.  Bring the sample volume up to **5 µl** with nuclease free H~2~O.

4.  Add **1 µl** of TOPO vector.

5.  Mix gently and quick spin.

6.  Incubate for 20 min at room temperature.

# Materials & 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 Carb 150 X-gal Plates                         Teknova                    L4940

                                                                              

  *[Reagents]{.underline}*                                                    

  ElectroMAX DH10B Cells                           Invitrogen                 18290015

  SOC Medium                                       Teknova                    S1640

  SOC Medium                                       Invitrogen                 Supplied with comp
                                                                              cells

  Glycerol                                         Sigma                      G-6279

                                                                              

  *[Equipment]{.underline}*                                                   

  Gene Pulser II                                   BioRad                     \-

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

# Procedure -- Transformation

> ***NOTE:** All reagents/stock solutions should be prepared prior to
> the start of the procedure.*

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

    -   Low range: 200

    -   High range: ∞ (not used)

    -   Capacitance: 25

    -   Voltage: 1.8 kV

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2.  **[Transformation]{.underline}**

    1.  Place on ice: one well-labeled Eppendorf tube and cuvette for
        each sample being transformed.

    2.  Thaw ElectroMax DH10B competent cells on ice (each tube contains
        100µl, enough for 2 reactions). Discard unused cells-DO NOT
        refreeze!

    3.  To the appropriately well-labeled, cold Eppendorf tube; add **1
        µl** of ligation product.

    4.  Once thawed, mix competent cells by swirling with pipette tip a
        few times.

    5.  Add **50 µl** eDH10B competent cells to the Eppendorf tube.

    6.  Mix by swirling the ligation and competent cells together with
        pipette tip a few times.

    7.  Transfer solution to the bottom groove of COLD cuvette and tap
        on tabletop a few times to settle solution to the bottom (must
        see even levels of cells on each side of the cuvette without
        bubbles).

    8.  Electroporate at 1.8 kV.

    9.  Transfer cell solution immediately to **950 µl** of SOC in a
        14ml falcon tube at room temperature (make sure SOC is clear,
        i.e. no growth).

> ***Important!** Transfer electroporation within 10 seconds to SOC.*
> Rinse cuvette with **50 µl** of the same SOC mixture to which you just
> added the cells.

10. Incubate within rotating wheel at 37^o^C for 1 hour.

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

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3.  **[Plating with Beads:]{.underline}**

    1.  Before transforming, prepare one well-labeled
        LB/CARB150/IPTG/X-gal agar plate per library by letting them
        warm to 37˚C in an incubator to dry agar down and open (\~ 90
        min).

    2.  After 60 minute transformation incubation, make a 10% glycerol
        transformation stock (**143 µl** 80% glycerol + full
        transformation = **\~ 1143 µl** glycerol transformation stock).

    3.  Cap, then mix transformation glycerol stock by inverting several
        times. \[Can store tube at -80˚C if not plating within 10 min.\]

    4.  Prepare a tube with **1.5 ml** of SOC (plating tube).

    5.  Add **10 μl** of the transformation glycerol stock to 1.5ml of
        SOC (plating tube).

    6.  Transfer remaining transformation glycerol stock into cryotube
        and store immediately at --80˚C for long term storage.

    7.  Mix tube by inverting several times (plating tube).

    8.  Place 10-15 beads onto bioassay.

    9.  Pipet entire volume of plating tube onto bioassay with beads
        (**\~1510 μl**). Pipet stock over the entire plate.

    10. Shake beads around on the bioassay until the stock has been
        evenly spread (Be Gentle!).

    11. Place bioassay on benchtop allowing stock to soak in completely.

    12. When bioassay is dry, pour beads off of bioassay into autoclave
        waste.

    13. Incubate the bioassays agar down in 37˚C incubator for 16-18
        hrs.

# Materials & Reagents -- QC PCR

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

  *[Disposables]{.underline}*                                                 

  96-well PCR plate                                USA Scientific             1402-9708

                                                                              

  *[Reagents]{.underline}*                                                    

  Taq DNA Polymerase                               Amersham Biosciences       27-0799-63

  10X PCR Buffer                                   Amersham Biosciences       \-

  10mM dNTP mix                                    MBI Fermentas              R0192

  pUC-F primer (CTTTACACTTTATGCTTCC)               IDT or Operon or your      \-
                                                   favorite                   

  pUC-R primer (GCAAGGCGATTAAGTTGG)                IDT or Operon or your      \-
                                                   favorite                   

  1 Kb DNA ladder                                  Invitrogen                 15615-016

                                                                              

  *[Equipment]{.underline}*                                                   

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

# Procedure -- QC PCR

# 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       27-0799-63

  10X PCR Buffer                                   Amersham Biosciences       \-

  10mM dNTP mix                                    MBI Fermentas              R0192

  pUC-F primer (CTTTACACTTTATGCTTCC)               IDT or Operon or your      \-
                                                   favorite                   

  pUC-R primer (GCAAGGCGATTAAGTTGG)                IDT or Operon or your      \-
                                                   favorite                   

                                                                              

  FailSafe PCR System (Enzyme mix for high GC)     Epicentre                  FS99250

  (choose 2x Buffer K)                             Epicentre                  FSP995K

                                                                              

  *[Equipment]{.underline}*                                                   

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

# Procedure -- PCR for QC of Library Insert Size

1.  **[PCR QC]{.underline}**

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

  ------------------------------------ ---------------------- ------------------------
                                       **[1X]{.underline}**   **[120X]{.underline}**

  Nuclease free H~2~O                  16.85 µl               2022.00 µl

  10X PCR Buffer                       2.00 µl                240.00 µl

  10mM dNTP (MBI)                      0.40 µl                48.00 µl

  pUC-F primer (10 pmol/µl)            0.28 µl                33.60 µl

  pUC-R primer (10 pmol/µl)            0.28 µl                33.60 µl

  Taq (Amersham)                       0.28 µl                33.60 µl

  Total Volume:                        **20.09 µl**           **2410.8 µl**
  ------------------------------------ ---------------------- ------------------------

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

3.  Spin down the plate.

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

5.  Using pipette tips only, pick 16-24 colonies per library into their
    own well. Be careful of your well location and of contamination. Mix
    tips in cocktail well.

6.  Quickly spin down plate.

7.  Set up a PE 9700 with the following colony PCR program (This is a 3
    hr 15 min protocol):

> 94 ^o^C -- 4 min.
>
> **94 ^o^C -- 30 sec.**
>
> **55 ^o^C -- 30 sec. 35 cycles**
>
> **68 ^o^C -- \*2 min.**
>
> 4^o^C -- Hold
>
> (\*modify with 5 sec. added on to each cycle for the 68 ^o^C
> elongation step.)

8.  Add **10µl** of 1X loading dye (must be faint so the PCR bands are
    not hidden) to PCR plate.

9.  Vortex & spin down plate to collect.

10. Load **15µl** of sample onto 1% agarose gel containing Ethidium
    Bromide (Carcinogen/Irritant) and save the remaining sample for
    later use if gel fails. Leave a well empty between libraries for
    loading **5µl** of marker 2.

11. Run for \~ 30 min at 120V.

12. Image gel and check for inserts at the appropriate size depending
    upon primer set used. **\~1.4 kb** (BAC16S), **\~1.4 kb** and/or
    **\~2.1 kb** (ARCH16S) and **\~0.7 kb** (EUK 18S).

13. Discard the gel in the appropriately labeled waste.

# Reagent/Stock Preparation

**[80% Glycerol Stock Solution]{.underline}**

40ml 100% Glycerol (pipette slowly)

10ml Nuclease-free H~2~0

Autoclave

Store at room temperature

**[6X Loading Dye]{.underline}**

75ml 100% glycerol

125ml Nuclease free H~2~0

0.05g Bromphenol Blue

0.05g Xylene Cyanole FF

Store at 4˚C

**[1X TAE Buffer]{.underline}**

To make 20 liters:

400ml 50X TAE Buffer

19.6L Milli-Q H~2~O

Store at room temperature

# Troubleshooting

**[(1) Only the positive control is visible.]{.underline}**

> This may indicate that the wrong concentration of the sample DNA was
> used for the reaction. The concentration may be too low or too high.
> Low DNA concentration may produce poor amplification, while too much
> template DNA may inhibit the amplification reaction due to the
> presence of PCR inhibitors in the sample. Amplification inhibition
> will be most commonly be the case.
>
> **[SUGGESTION:]{.underline}**
>
> Run template DNA serial dilutions as follows.

**[Dilutions:]{.underline}**

> 1/10 dilution: **1 µl** of stock DNA to **9 µl** of nuclease free
> H~2~O
>
> 1/100 dilution: **1 µl** of (1/10 dilution) to **9 µl** of nuclease
> free H~2~O
>
> 1/1000 dilution: **1 µl** of (1/100 dilution) to **9 µl** of nuclease
> free H~2~O
>
> Use **1 µl** from the above dilutions for amplification reaction.

**1 µl** of each dilution for bacterial 16S rRNA gene amplification

**1 µl** of each dilution for archaeal 16S rRNA gene amplification

**1 µl** of each dilution for eukaryotic 18S rRNA gene amplification

Bring volume up to **18.9 µl** with nuclease free H~2~O

Dispense **26.1 µl** of the appropriate PCR master mix for each primer
combination.

> **NOTE:** *For environmental samples, using multi-channel pipettor
> transfer 2 times **15 µl** / sample into rows below.*
>
> **NOTE:** *When the above method still fails to amplify, the given
> gene of interest and thus superkingdom may not be present in the
> sample.*

**[(2) Both negative and positive controls are visible.]{.underline}**

This indicates contamination.

> **[SUGGESTION:]{.underline}**

Toss all amplified products and repeat 16S/18S rRNA gene PCR
amplification.

> **NOTE:** *Be careful when dispensing samples to ensure no cross
> contamination.*

**[(3) Multiple bands of different sizes are visible.]{.underline}**

> This may indicate that the (environmental) sample contains organisms
> with rRNA genes of different lengths. Archaeal 16S rRNA genes as well
> as eukaryotic 18S rRNA genes may contain introns of varying size.
>
> Moreover, unspecific amplification may occur.
>
> **[SUGGESTIONS:]{.underline}**
>
> Repeat amplification, run a positive, negative control, and run 1ul of
> undiluted DNA. In addition to adding positive and negative controls,
> **1 µl** of DNA will be added as a DNA quality control. If multiple
> bands do not appear in the 1ul DNA sample, then the library should be
> cloned and handed off for sequencing.
>
> **NOTE:** *This will help determine if the DNA was affected by the
> heating and cooling in the thermocycler, causing multiple band
> patterns.*

Add **1 µl** of DNA sample to a clean well

Bring volume up to **45 µl** with of nuclease free H~2~O

Do not add any PCR master mix to the sample.

The rRNA Gene library PCR QC show different band sizes.

> It is perfectly normal for ARCH 16S and EUK 18S libraries to have
> different size PCR product.

# SOP Approval

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

  **Lab Supervisor**                              

  **Research &                                    
  Development**                                   

  **Instrumentation**                             

  **QC**                                          

  **Purchasing**                                  

  **EH & S**                                      

  **Informatics**                                 

  **Seq Assessment &                              
  Analysis**                                      

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

# Appendix

**[AUDIT TRACKING]{.underline}**

**[PROCEDURAL CHANGES]{.underline}**