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From 2011.igem.org

July 24th - July 30th

Sunday, July 24

Monday, July 25

Tuesday, July 26

Lab work done by: Charlie Chung, Nicholas Kramer, Jim Mathew

Objectives

To anneal primers containing the AviTag and use as inserts in ligation reactions

To set up various ligation constructs

Phosphorylation of Primers

5μL 10x T4 DNA ligase buffer

3μL primer

1μL T4 polynucleotide kinase

41μL H2O

50μL Total

• Incubate at 37°C for 30 minutes

Heat Inactivation of Polynucleotide Kinase

• Incubate at 65°C for 20 minutes

Primer Annealing

• Incubate forward and reverse primer in a 1:1 ratio at 95°C for 3 minutes
• Let cool to room temperature for ~10 minutes

Can also use "Anneal" program on thermocycler

Ligation

• VioE + AviTag + Backbone

8.8μL pZE12 vector backbone (cut with KpnI and ClaI)

4.2μL VioE

2.4μL H2O

2.0μL T4 DNA ligase buffer

0.8μL Primer Pair A

0.8μL Primer Pair B

1.0μL T4 DNA ligase

20μL Total

• GFP + AviTag + Backbone

9.4μL H2O

3.6μL pZE12 vector backbone (cut with KpnI and ClaI)

2.4μL GFP

2.0μL T4 DNA ligase buffer

0.8μL Primer Pair A

0.8μL Primer Pair B

1.0μL T4 DNA ligase

20μL Total

• RFP + AviTag + Backbone

10.8μL H2O

4.6μL RFP

3.6μL pZE12 vector backbone (cut with KpnI and ClaI)

2.0μL T4 DNA ligase buffer

0.8μL Primer Pair A

0.8μL Primer Pair B

1.0μL T4 DNA ligase

20μL Total

• GFP + Backbone

13μL pZE12 vector backbone (cut with KpnI and SphI)

2.4μL GFP

2.0μL T4 DNA ligase buffer

1.6μL H2O

1.0μL T4 DNA ligase

20μL Total

• AviTag + Backbone

7.8μL pZE12 vector backbone (cut with SphI and ClaI)

7.6μL H2O

2.0μL T4 DNA ligase buffer

0.8μL Primer Pair A

0.8μL Primer Pair B

1.0μL T4 DNA ligase

20μL Total

In all above reactions, volume of plasmid vector added corresponds with 100ng backbone

Prepare control ligation reactions (no inserts) for each of the above constructs

• Same volume of vector backbone, buffer, ligase
• Volumes of all inserts (i.e. gene or primer pairs) go toward volume of H2O

After ligation setup, incubate in 16°C water bath overnight

Wednesday, July 27

Lab work done by: Youjin Cho, Charlie Chung

• De-salted the ligation samples that were set overnight and transformed them into DH5α electrocompetent cells with ampicillin resistance

Samples

AviTag + pZE12 (backbone)

VioE + AviTag + pZE12

GFP + AviTag + PZE12

RFP + AviTag + pZE12

GFP + pZE12

pZE12 control

VioE control

AviTag control

Thursday, July 28

Lab work done by: Youjin Cho, Bill Jo, Nancy Li

• Miniprep of transformed ligation products
• Submit for sequencing

Sequencing Order #: 10253750

Samples

AviTag + pZE12-1

AviTag + pZE12-2

AviTag + pZE12-3

VioE + AviTag + pZE12-1

VioE + AviTag + pZE12-2

VioE + AviTag + pZE12-3

GFP + AviTag + PZE12-1

GFP + AviTag + pZE12-2

RFP + AviTag + pZE12-1

RFP + AviTag + pZE12-2

GFP + pZE12

Friday, July 29

Morning lab work done by: Youjin Cho, James Mathew

• Set up digestion reaction for VioE, pZE12 (backbone) cut with KpnI and SphI

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Afternoon lab work done by: James Mathew, Charlie Chung

Objective

• Ligate the digested gene for the VioE enzyme (now with the appropriate sticky ends) onto the pZE12 vector backbone for future AviTag insertion
• Interpret sequencing results and plan out next steps accordingly

NanoDrop Concentrations of Miniprep Purification Products

• 19.7ng/μL = pZE12 vector backbone cut with KpnI-HF and SphI-HF (HF = high fidelity)
• 26.8ng/μL = VioE gene cut with KpnI-HF and SphI-HF

(VioE + Backbone) Ligation Reaction Mixture

7.6μL pZE12 vector backbone cut with KpnI-HF and SphI-HF

5.9μL H2O

3.5μL VioE gene (insert)

2.0μL 10x T4 DNA ligase buffer

1.0μL T4 DNA ligase

20μL Total

• Incubate in 16°C water bath overnight.
• NOTE: Forgot to treat pZE12 vector backbone with calf intestinal alkaline phosphatase (CIAP) for removal of 5' phosphate groups on the plasmid. Because noisy background of self-ligated pZE12 (without VioE gene insert) will be likely, will probably ignore the ligation product.

Sequencing Results

• The nucleotide sequence of (GFP + Backbone) & (AviTag + Backbone) ligation constructs indicate that GFP and AviTag have independently been inserted into the pZE12 plasmid backbone.
• Peculiar that the rest of the ligation constructs returned "noisy or failed" sequencing results because all the plates of bacteria transformed with the ligation product are at least two times more confluent than their corresponding control plates.

Pick Colonies & Bacterial Culture

• Transferred the successful colony of (AviTag + Backbone) to a 25mL culture flask (incubating at 37°C in Room 304)
• Despite a noisy sequencing result of (VioE + AviTag + Backbone), the plate shows significantly more growth than its control plate.
• Picked three colonies and transferred to individual 5mL culture tubes (incubating at 37°C in Room 304)

Saturday, July 30

Lab work done by: Deborah Liu, Charlie Chung

• Miniprep purification of the three 5mL cultures of (VioE + AviTag + pZE12)

- NanoDrop spectrophotometry to determine DNA concentration

• Miniprep purification of the 25mL culture of (AviTag + pZE12) into two Eppendorfs

- NanoDrop spectrophotometry to determine DNA concentration

• Followed the standard Qiagen Miniprep protocol