iGEM 2010 — Team Georgia_State

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# Team:Georgia State/Notebook

### From 2010.igem.org

![](https://static.igem.org/mediawiki/2010/f/f9/Atlanta_Skyline_at_Sunset_Georgia.png)

* [Home](https://2010.igem.org/Team%3AGeorgia_State)
* [Project](https://2010.igem.org/Team%3AGeorgia_State/Project)
  + [About The Toolkit](https://2010.igem.org/Team%3AGeorgia_State/Aboutthetoolbox)
  + [Oursystem](https://2010.igem.org/Team%3AGeorgia_State/Oursystem)
  + [Producing The Antigen](https://2010.igem.org/Team%3AGeorgia_State/Producingtheantigen)
  + [Future Applications](https://2010.igem.org/Team%3AGeorgia_State/Futureapplications)
  + [Safety](https://2010.igem.org/Team%3AGeorgia_State/Safety)
* [Toolkit](https://2010.igem.org/Team%3AGeorgia_State/Parts)
* [Why Pichia](https://2010.igem.org/Team%3AGeorgia_State/WhyPichia)
  + [Pichia Vs Saccharomyces](https://2010.igem.org/Team%3AGeorgia_State/WhyPichia#Pichia_Vs_Saccharomyces)
  + [Easy and Inexpensive to Culture](https://2010.igem.org/Team%3AGeorgia_State/WhyPichia#Easy_and_Inexpensive_to_Culture)
  + [High production of foreign Protein while low levels of endogenous protein](https://2010.igem.org/Team%3AGeorgia_State/WhyPichia#High_production_of_foreign_Protein_while_low_levels_of_endogenous_protein)
  + [Pichia pastoris versus Esherichia coli](https://2010.igem.org/Team%3AGeorgia_State/WhyPichia#Pichia_pastoris_versus_Esherichia_coli)
* [Protocols](https://2010.igem.org/Team%3AGeorgia_State/Protocols)
  + [Preparation Of Electrocompetent Cells](./)
  + [Preparation of Heat Shock Competent Cells](./)
  + [Transformation Using Electroporation](./)
  + [Transformation Using Heat Shock](./)
* [The Team](https://2010.igem.org/Team%3AGeorgia_State/Team)
* [Notebook](https://2010.igem.org/Team%3AGeorgia_State/Notebook)

|  |
| --- |
| Contents   * [1 July 1, 2010](#July_1.2C_2010) * [2 July 2, 2010](#July_2.2C_2010) * [3 July 6, 2010](#July_6.2C_2010) * [4 July 7, 2010](#July_7.2C_2010) * [5 July 8, 2010](#July_8.2C_2010) * [6 July 13, 2010](#July_13.2C_2010) * [7 July 14, 2010](#July_14.2C_2010) * [8 July 15, 2010](#July_15.2C_2010) * [9 July 20, 2010](#July_20.2C_2010) * [10 July 22, 2010](#July_22.2C_2010) * [11 August 3, 2010](#August_3.2C_2010) * [12 August 4, 2010](#August_4.2C_2010) * [13 August 5, 2010](#August_5.2C_2010) * [14 August 8, 2010](#August_8.2C_2010) * [15 August 8, 2010](#August_8.2C_2010_2) * [16 August 9, 2010](#August_9.2C_2010) * [17 August 12, 2010](#August_12.2C_2010) * [18 September 2nd, 2010](#September_2nd.2C_2010) * [19 September 7th, 2010](#September_7th.2C_2010) * [20 September 10th, 2010](#September_10th.2C_2010) * [21 September 13th, 2010](#September_13th.2C_2010) * [22 September 21st, 2010](#September_21st.2C_2010) * [23 September 22nd, 2010](#September_22nd.2C_2010) * [24 September 22nd, 2010](#September_22nd.2C_2010_2) * [25 September 23rd, 2010](#September_23rd.2C_2010) * [26 September 23rd, 2010](#September_23rd.2C_2010_2) * [27 September 23rd, 2010](#September_23rd.2C_2010_3) * [28 September 27th, 2010](#September_27th.2C_2010) * [29 October 2nd, 2010](#October_2nd.2C_2010) * [30 October 4th, 2010](#October_4th.2C_2010) * [31 October 5th, 2010](#October_5th.2C_2010) * [32 October 18th, 2010](#October_18th.2C_2010) * [33 October 20th, 2010](#October_20th.2C_2010) |

## **July 1, 2010**

Joe

Plated transformed cells containing either GFP (control) or iGEM part onto LB plates with 10µg/mL ampicillin (2 plates each, 10mL or 100mL)
Also plated ≈100mL (remaining amount) of cells onto LB plates not containing ampicillin.
Spread plates with hockey stick and placed in 37°C at 7:35.

## **July 2, 2010**

Alykhan and Virginia
DNA purification and ligation
Replated E. coli culture

## **July 6, 2010**

Dan

Replated E. coli culture

Alykhan

Transformation 9A, 8I (digested) & GFP.

## **July 7, 2010**

Joe, Kendra, Angie

Replated colonies from 9A RFP plate (3 plates).
Colonies on broth 100 µL for 9A but no fluorescence observed
9A survivor colonies replated onto 10 µg/mL ampicillin plates and incubated at 30°C
Mother plate in fridge
Made 1000mL YPD
Growing P. anomala in YPD broth for competent cells protocol

## **July 8, 2010**

Angie, Kendra, Melissa, Nishedh, Alykhan

Diluted pichia cells in a volume of 50mL to a OD600 = .186+.201
Transform pars 12E and 12M into E. coli

## **July 13, 2010**

Virginia
10 glycerol stocks of each

```
         o P. pastoris
         o 9A E. coli
```

Inoculated 12E broth for plasmid extract and glycerol stocks
Plate of P. pastoris for quality control

## **July 14, 2010**

Virginia, Joe, Angie, Alykhan

10 glycerol stocks of 12E
P. pastoris competent cells
Started, ready at 2pm
Plasmid resuspension buffer made
LB agar aliquots

```
         o warm in water bath
         o one 10mL tube for 1L agar
```

extraction of 12E+9A plasmid parts: white tubes in freezer

## **July 15, 2010**

Virginia, Angie, Alykhan, Joe

Cut plasmid and ligated
P. pastoris cells ready in -80°C

## **July 20, 2010**

Melissa, Alykhan

Prepared tris HCl for buffer solutions

## **July 22, 2010**

Virginia

Prep LB, YPD plate solution
Prep NEB 2+3 buffer solutions

## **August 3, 2010**

Joe, Virginia

Tried to extract 12E plasmid with Jetgene kit starting with cells stored in resuspension buffer. (Started with step 2). Cell solution was very viscous. Difficult to pipette. Did not form a pellet in step 4.
Inoculated LB+Ampicillin with 12E and stored at 37°C from glycerol stock in -80°C.

```
         o Will try again with fresh cells
         o Ran out of buffer in Jetgene kit.
```

## **August 4, 2010**

Virginia 12E plasmid extraction from 5-min kit. Lysis solution and wash buffer taken from eppendorf kit needs to be confirmed on Nano Drop.

## **August 5, 2010**

12E SpeI protocol, PstI protocol
9A PstI proocol
9A was purified as the Xba I previously used on it was not fast digest
Gel extraction of 9A and 12E

## **August 8, 2010**

Joe

Prepared YPD+sorbitol

```
         o 10g yeast extract
         o 20g peptone
         o 182.2g sorbitol
         o 900ml H20
```

Inoculated 250ml flash of YPD with P. pastoris from 6-29-10 culture in SAB

```
         o added 500ml of SAB culture into ≈100-125ml YPD, stored in shaker in 30°C incubator
```

## **August 8, 2010**

Centrifuge P. pastoris to prep for electroporation
Competent cells prepared, ready to pulse
Transformed/electroporated P. pastoris with 9A and GFP

## **August 9, 2010**

Joe

Prepared YPD/Hepes
Prepared 10mL of DTT
Inoculated 2 1L flasks containing 250mL YPD with 500mL of P. pastoris from overnight culture. Stored in -80°C room at 30°C in shaker.
Protocol modifications for P. pastoris transformation are in notebook

## **August 12, 2010**

```
Reading about P. pastoris as vaccine vector
```

Started engineering of plasmid

Joe

Inoculated LB+Ampicillin broth with glycerol stock of E. coli containing GFP

```
Located in 37°C incubator in Crow lab.
```

## **September 2nd, 2010**

```
Melissa,  Joe
```

Inoculated LB broth with 250 uL 12E
100 mL freshly-made LB broth
+ 1 mL LB + 10x ampicillin
~100 mL 1x ampicillin + LB broth
12E placed in shake flask @ 37°C

## **September 7th, 2010**

Alykhan, Nishant, Bhavik, Kathy, Nishedh
Innoculated 12E (arabinose-induced promoter)
Plates put in 37°C incubator for 14 hours
P. pastoris from -80°C inoculated in LN broth and plated. Grown for 2 days.
DNA extracted and placed in -20°C freezer [PCR needs to be done using primers]
6G (Lac regulating promoter) was transformed into E. coli
Competent cells were used from -20°C freezer
Remainder of DNA was placed in -20°C freezer
Transformation was done in duplicate and plates were put in 37°C incubator for 14 hours
Growing part was transformed to broth and a new plate [parts must be ligated; DNA must be extracted]

## **September 10th, 2010**

Joe
Prepared 1x working stock of Amro’s Cox 5-R (Ref# 49115848) and Cox 5-F (Ref# 49115847) to use as positive control for PCR reactions
90 µL ddH2O + 10 µL 10x stock = 100 µL 1x working stock

## **September 13th, 2010**

Joe
“Isolation of Histidinol Dehydrogenase from Pichia pastoris using PCR”
Two sets of primers were designed based on previously published primers but modified to add EcorI and SpeI sites
Set I: F=0848 R=0849
Set 2: F=0850 R=0851
Cont: F=Cox5 R=Cox5
Used following ratios: 2.5 µL MasterMix, 1 µL F. primer, 1 µL R. primer, 1 µL DNA (45 mg/mL), 9.5 µL water. Total volume 25 µL in PCR reaction tubes.
PCR settings: adjusted 74 time from 30 sec → 3:00 min
Reaction:
F R
Control Cox5 Cox5
Set 1 0848 0841
Set 2 0850 0851
Hybrid 1 0848 0851
Hybrid 2 0850 0849

Tube labels: Con, 1, 2, 3, 4. Started at 3:00 PM, finished at 5:35 PM.

## **September 21st, 2010**

“Insertion of His4 PCR product into plasmid”
Purpose: to insert the PCR produce of F=0850 and R=0851 into the RFP plasmid
Procedure: (PCR)
9A (RFP) digest with SpeI and EcoRI: 15 µL nuclease-free H20, 2 µL fast digest 10x buffer, 2 µL DNA, 1 µL each enzyme. Total volume=21 µL
H (histidinol) digest with SpeI and EcoRI: 17 µL nuclease-free H20, 2 µL fast digest, 10 µL DNA, 1 µL each enzyme. Total volume=31 µL
Heated at 37°C 5 min
Purification of plasmid and His4: used QIA quick PCR purification protocol
Plasmid: Total volume from RE digest = 20 µL. 20 µL plasmid + 100 µL PBI buffer (5x PBI) = 120 µL
H: total volume = 30 µL. 30 µL + 150 µL PBI buffer
Gel:
\_\_\_\_\_\_\_ \_\_\_\_\_\_\_ \_\_\_\_\_\_\_ \_\_\_\_\_\_\_ \_\_\_\_\_\_\_ \_\_\_\_\_\_\_ \_\_\_\_\_\_\_
L 9A \_\_\_\_\_\_\_ P L
A 9A U A
D C D
D D
E E
R R

Extracted and plated with LB on agar. Poor plates.
Discussion: This experiment failed at some point as noted by the lack of bands after running a gel to isolate the 9A plasmid backbone. The successful banding patterns by Kristin’s PUC plasmid indicate that the error occurred in either the restriction digest or during the purification process. It is most likely that the problem was in the restriction digest. The enzymes used were fast-digest enzymes and were not inactivated after the 5 min incubation. They were placed at room temperature for approximately an hour before being purified. It is believed that the enzymes continued to digest the DNA to completion. Future attempts should require a quicker transition time from the digest, an inactivation step, and potential postponing of the purification step until after the ligation has been performed.

## **September 22nd, 2010**

“PCR with primers 0850 and 0851”
The purpose is to obtain His4 fragment from P. pastoris DNA. This reaction was performed successfully on September 10th , 2010.
P. pastoris DNA 580 ng/µL. 1:10 dilution → 58 ng
12.5 µL MasterMix 2x, 1 µL F. primer, 1 µL R. primer, 1 µL DNA (58 ng/µL), 9.5 µL H2O)

## **September 22nd, 2010**

“Insertion fo His4 PCR product into 9A plasmid backbone”
The purpose is to ligate PCR product of F=0850 and R=0851 into RFP (9A) plasmic backbone. The process involves digesting both components with EcoRI and SpeI, running a gel to remove RFP, and ligation.
Procedure:
1. Digest
a. . 9A digest: 15 µL nuclease-free H2O, 2 µL fast-digest 10x buffer, 2 µL DNA, 1 µL of each enzyme. Total volume is 21 µL.
b. His4 digest (PCR fragment): 17 µL nuclease-free H2O, 2 µL fast-digest 10x buffer, 10 µL S3 (his4), 1 µL each enzyme. Total volume is 31 µL.
Inactivate at 80°C for 5 min in thermocycler
2. Purification of His4 fragment
a. Add 5 volumes of buffer PBI to 1 volume sample
i. 150 µL PBI to 30 µL sample
b. Place into a QIA quick column inside a 2 mL collection tube
c. Centrifuge 60 s and discard flowthrough
d. Add .75 mL PE to column
i. Centrifuge 60 s and discard flowthrough
e. Centrifuge again for 6 seconds
f. Place column in clean centrifuge, add 50 µL EB buffer, and spin 60 s to elute DNA
g. Place on ice in centrifuge tube

3. Gel excision of 9A plasmid backbone
a. Also ran PCR products from 0850 and 0851 from today and 9-10-10
C RFP H1 H2
\_\_\_\_\_\_ \_\_\_\_\_\_ \_\_\_\_\_\_ \_\_\_\_\_\_ \_\_\_\_\_\_ \_\_\_\_\_\_ \_\_\_\_\_\_ \_\_\_\_\_\_
L L
A \_\_\_\_\_\_ A
D \_\_\_\_\_\_ \_\_\_\_\_\_ D
D D
E \_\_\_\_\_\_ E
R \_\_\_\_\_\_ R

Cut out 3 gels segments: RFP, backbone, H.S.
Weights: 0.30 g plasmid backbone, 0.15 g RFP, 0.40 g His4
Add 3 volumes of Buffer QG to 1 volume of gel (100 mg~100 µg)
Plasmid backbone = 300 mg → 900 µL QG
RFP = 150 mg → 450 µL QG
His4 = 400 mg → 1200 µL
Incubate for 10 min @ 50°C. Shake periodically to help dissolve gel.
Add one part isopropanol and mix
PB → +300 µL
RFP → +150 µL
His4 → +400 µL
Apply to spin column (800 µL of sample at a time). Centrifuge for 1 min.
Add .5 mL QG to column and spin 1 min
Wash with .75 mL PE. Centrifuge 1 min.
Discard flow-through. Spin again for 1 min.
Elute DNA by placing column into clean centrifuge tube and applying 50 µL EB buffer

## **September 23rd, 2010**

9A backbone with sticky ends. Ligate with His4 after digestion (EcoRI and SpeI)
His4 → 11.7 ng/ µL
Plasmid backbone → 8.3 ng/µL
Ligation: used 3 µL of 8 ng/µL plasmid backbone → 24 ng; and 41 µL of 11 ng/ µL His4 → 44 ng
Incubated with thermal cucler for 4 hours at 16°C, 10 min @ 70°C, and hold at 4°C overnight
Ligation: Used 1 µL 10x ligase bugger, 0.5 µL mM DDT, 1 µL 10 mM ATP, 3 mL plasmid backbone, 10 µL ligase dilution buffer, 4 µL His4

## **September 23rd, 2010**

“Isolation of pAOX1 and pGAP”
A → pAOX1
B→ pGAP
C→Control (cytochrome from Dr. Amro)
Master Mix 12.5 µL
Forward primer 1 µL
Reverse primer 1 µL
Pichia DNA 1 µL
Water 9.5 µL

## **September 23rd, 2010**

Bianca
Ligation
Add these in a PCR tube:
1. 1 µL 10x ligase buffer
2. 0.5 µL 100 mM DDT
3. 1 µL 10 mM ATP
4. 3 µL plasmid backbone
5. 1 µL DNA ligase
6. 4 µL His4
Total volume is 10 µL
1 µL (0.1 TH DNA ligase + 0.9 DNA ligase buffer)
In thermocycler @ 6:26

## **September 27th, 2010**

Two LH and two LR lanes run at 100 volts, 400 mA, 60 minutes.
Gel: 2.5 µL brotium gel red, 0.43 g agarose, 35 mL TAE

## **October 2nd, 2010**

Alykhan
The ligated RFP and plasmid backbone and the part (His-4) were run in a gel to check the ligation.
1,2% agarose gel was made (0.9 g in 75 mL). 7.5 µL of biotine red was added
Lane 1: ladder; 2: RFP; 3: RFP; 4: Part 1; 5: Part 2; 6: ladder
10 µL of sample and 2 µL of formaldehyde dye was loaded. 6 µL of ladder was loaded. Gel was run for one hour at 100 volts
Excision:
1st: 0.78 g → 0.89 g
0.11 (110 mg)
2nd: 0.80 → 0.88
80 mg
Add buffer: 330 µL to 1st and 80 µL to 2nd
After dissolves: add 110 µL isopropenol
Sample 1: 7.1
Sample 2: 7.4

## **October 4th, 2010**

Joe
“Isolation of pGAP, pAOX1, FLD1”
The purpose of this experiment is to isolate the parts pGAP, pAOX1, and FLD1 from DNA of P. pastoris through PCR rxn. 2 sets of primers have been designed for each part. The first set of each contains an Xba site on the forward primer and a Spe1 site on the reverse. The second set of primers adds EcoRI, Not1, and Xba to the forward and Spe1, Not !, and Pst onto the reverse primer. The second product of the second set will yield a biobrick product, ready for insertion into plasmid \_\_\_\_\_\_\_. However, because adding 3 restriction enzyme sties decreases primer quality, hybrid reactions will be run to increase likelihood of obtaining a useable product.
Methods
Tube # FLD1
F1 (F) 8286 (R) 8287
F2 (F) 8286 (R) 8289
F3 (F) 8288 (R) 8287
F4 (F) 8288 (R) 8289
Product: X—S, X—SNP, ENX---S, ENX---SNP → high temp
pGAP
G1 (F) 1148 (R) 1149
G2 (F) 1148 (R) 8285
G3 (F) 8284 (R) 1149
G4 (F) 8284 (R) 8285
X---S, X---SNP, ENX---S, ENX---SNP → high temp
pAOX1
A1 (F) 1146 (R) 1147
A2 (F) 1146 (R) 8283
A3 (F) 8282 (R) 1147
A4 (F) 8282 (R) 8283
X---S, X---SNP, ENX---S, SNX---SNP → high temp
For each reaction, the following ratio was used: 12.5 µL MasterMix, 1 µL forward primer, 1 µL reverse primer, 1 µL 58 ng/µL Pichia DNA, 9.5 µL H2O. 25 µL total.
[ran gel]
F: 1.3 Kb (contains EcoRI site! Must use different enzyme!)
A: .9 Kb (Gel showed successful amplification of all samples except F1)
G: .5 Kb
For isolating pGAP, the FR sample showed the strongest bands. However, it was inaccessible from extraction.

## **October 5th, 2010**

Nishedh and Bhavik
Restriction digest of the parts. Cut the A4, G4 with EcoRI and PstI. Reaction volumes:
A4: 10 µL water, 2 µL 10x buffer, 17 µL DNA, 1 µL EcoRI, 1 µL Pst1. Total 31 µL. 5 min at 37 degrees C.
G4: 17 µL water, 2 µL 10x buffer, 10 µL DNA, 1 µL EcoRI, 1 µL PstI. Total 31 µL. 5 min at 70 degrees C
Since F4 has an EcoRI site in it, we will use a different approach. Cut F4 with X-ba1 and PstI
First we have to cut both with Xba1: Reaction volume:
F4: 10 µL DNA, 18 µL water, 2 µL 10x buffer, 2 µL Xba1 = 32 µL.
RFP: 5 µL DNA, 23 µL water, 2 µL 10x, 2 µL Xba1: 32 µL.
Overnight in the thermocycler.
Purification of A4, G
31 µL x 5 = 155 µL PBI spin
750 µL PE. Spin.
Spin again.
50 µL of EB spoin.
Quantify
A4 → 9.5 ng/µL
G4 → 5.7 ng/ µL
Ligation
Mix all these together in PCR tubes: 1 µL 10x ligase buffer, 0.5 µL 100 mM DDT, 1 µL of 10 mM ATP, 1 µL plasmid vector PCBIA3, 10 µL DNA, 0.9 µL ligase dilution buffer, 0.1 µL T4 DNA ligase, 0.5 µL nuclease-free water. Total: 15 µL. Put in thermocycler.
Purify F & R 20 mins. Fast digest with PstI 10 min. Ligate F4 and R in vector.

Isolation of pTEF from PCR
The purpose of this experiment is to isolate the pTEF promoter from pichia pastoris DNA. Two sets of primers were used for this experiment to produce PCR products with different restriction sites.
The primers used were stored in the -20° C freezer.
Set 1 Sites Added
Forward 3414 X----
Reverse 3415 ----S

Set 2 Sites Added
Forward 9201 ENX---
Reverse 9202 ---SNP

PCR
12.5 µL Mastermix
1 µL Forward
1 µL Reverse
1 µL Protein DNA (58 ng/µL)
9.5 µL double sterile water
PCR Rxn
Tube # Forward Primer Reverse Primer Product
1 3414 3415 X----S
2 3414 9202 X----SNP
3 9201 3415 ENX----S
4 9201 9201 ENX----SNP

Modifications:
- Samples 1-3 were modified from 55°C →57°C.
- Sample 4: run on IGEM high temp cycle. Modified from 66°C→63°C.

How to Prepare a Gel

Need:
1.2% agarose and .001 % Bio-red
Examples
35 ml gel → 0.42 g Agarose
35 ml 1X TAE
3.5 µL Bio-Red
Or
75 ml gel → 0.9 g Agarose
75 ml 1X TAE
7.5 µL H₂O
1. Add weighed amount of agarose to flask
2. Add 1X TAE
3. Microwave for ~60 seconds, stirring occasionally
4. Prepare gel tray with desired wells in place
5. Add Bio-Red after microwaving, swirl to mix, and pour into gel tray.
6. Let solidify for at least 30 minutes.
7. Remove wells.

Gel Electroporation
A₄ & G₄ → The ligated parts were ran in a gel to check if the ligation was successful.
The purified % digested F₄ and RFP were ran on a gel to check if they were successful.
Ladder F₄ A₄ G₄ RFP Ladder
- None -
- -
- - -
-
-

Ran at 120 V, 200 mA, for 60 minutes.
Results:
Two bands were located in the RFP lane
Indicates:
1- RFP (part)
2- Plasmid backbone.
A₄ & G₄-
-1 band in each lane around 3000 base pairs. This could possibly indicate that the past was
ligated into the plasmid backbone.

Transformation
A₄ &G₄ were transformed using the heat shock protocol.
Results:
- No growth on LB with ampicillin plates for either part.
- LB plates displayed viability.
- Primers needed to be made to amplify the ligated part because the gel reveals evidence of ligation, but transformation not successful.

## **October 18th, 2010**

“Transformation of TOP10 cells”
Melissa
TOP10 cells were transformed (TOP10 protocol) with alcohol oxidase, glyceraldehyde-3-phosphate, and RFP as a control. Goal is to determine optimal media for growth.
2 µL of each were added to 50 µL vials of competent cells. Incubated on ice 30 min. Heat shock at 42 degrees C 30 sec. 250 µL SOC beroth added to each. Vials centrifuged at 37 degrees 1 hour. Plated. 20 µL each on LB plated with ampicillin. 700 µL each on LB plated. 100 µL each on YPD as control. Plates inverted and incubated at 37 degrees 14 hours.

Gel electrophoresis:
90 A4 – 0.09: 0.80 – 0.89 (6.0)
220 F4 – 02.2: 0.75 – 0.98 (9.3)
230 G2 – 0.23: 0.82 – 1.05 (9.4)
300 T1 – 0.30: 0.81 – 1.05 (9.0)
QG
A4 → 270 µL
F4 → 660 µL
G2 → 690 µL
T1 → 900 µL

## **October 20th, 2010**

Angie
Digestion: G1 and F digest with Pst1
17 µL DNA, 10 µL water, 2 µL 10x buffer, 1 µL Pst1 = 30 µL
Purify using PCR purification kit.

[Team](/Team%3AGeorgia_State/Team "Team:Georgia State/Team") |
[Official Team Profile](https://igem.org/Team.cgi?year=2010&team_name=Georgia_State) |
[Project](/Team%3AGeorgia_State/Project "Team:Georgia State/Project") |
[Parts Submitted to the Registry](/Team%3AGeorgia_State/Parts "Team:Georgia State/Parts") |
[Modeling](/Team%3AGeorgia_State/Modeling "Team:Georgia State/Modeling") |
**Notebook** |
[Safety](/Team%3AGeorgia_State/Safety "Team:Georgia State/Safety")

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