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Team:Cornell/Week 12
From 2011.igem.org
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Revision as of 01:34, 24 August 2011
Week 1 | Week 2 - 5 | Week 6 | Week 7 | Week 8 | Week 9 | Week 10 | Week 11 | Week 12 | Week 13 | Week 14 | Week 15 | Week 16 | Week 17 | Week 18 | Week 19 | Week 20 | Week 21 |
August 21st - August 27th
Sunday, August 21
Evening lab work done by: Youjin Cho, Charlie Chung
- Picking Colonies and Starting Up Cultures of vioB Ligation Reaction
- (Avi-Tagged pZE12 backbone) control plate shows no growth of colonies
- (vioB + Avi-Tagged pZE12 backbone) plate shows numerous colonies
- - Picked three colonies (vioB+Avi+BB #1, 2, 3) and started 3mL cultures in LB with 3μL ampicillin
- - Incubated overnight in the 37°C shaker of Room 304
- PCR Reaction Setup of GFP (because sequencing results were negative)
- 38.5μL ddH2O
- 5μL 10x buffer
- 2.5μL dNTP
- 1μL forward GFP primer
- 1μL reverse GFP primer
- 1μL (pZE12+GFP) backbone template
- 1μL Vent DNA polymerase
- 50μL Total
- Annealing temperature of 54.2°C
- 1 minute extension cycle
Monday, August 22
Morning lab work done by: Youjin Cho
- Objective
- Miniprep the vioB+Avi-Tag+pZE12 samples and submit for sequencing.
- Miniprep and Sequencing
- Miniprepped the vioB+avi-tag+pZE12 (1,2,3) samples using standard Qiagen Miniprep protocol.
- Using 1μL of pZE12 reverse primer dilution and 17μL of each template, prepared and submitted the samples for sequencing.
- Order number: 10255430
Tuesday, August 23
Afternoon lab work done by: Claire Paduano, Maneesh Gupta, Youjin Cho, Charlie Chung
- Objective
- Set up new ligation of GFP and Avi-Tagged backbone
- Send out gel purified GFP PCR reaction product to troubleshoot why our procedure isn't working
- Conducted gel electrophoresis on PCR reaction product of GFP
- - Visualization of migrated DNA fragments does not show a GFP band around 750bp
- Redid PCR on GFP
- - Note: This time, we made sure to add 1µL MgSO4
- 37.5μL ddH2O
- 5μL 10x buffer
- 1μL 25mM dNTP
- 1μL MgSO4
- 1μL forward GFP primer
- 1μL reverse GFP primer
- 1μL (pZE12+GFP) backbone template
- 1μL Vent DNA polymerase
- 48.5μL Total
- Annealing temperature of 54.2°C
- 1 minute extension cycle
- Gel electrophoresis
- -Visualization shows GFP band around 750bp
- Gel purified GFP product
- -Visualization shows GFP band around 750bp
- Digestion Setups
- GFP insert
- 36μL H2O
- 6.5μL GFP (1μg)
- 5μL 10x NEBuffer 4
- 0.5μL 100x BSA
- 1.0μL KpnI-HF
- 1μL SphI-HF
- 50μL Total
- Avi-Tagged pZE12 vector backbone
- 32.9μL H2O
- 9.6μL VioE in Avi-Tagged pZE12 vector backbone (1μg)
- 5μL 10x NEBuffer 4
- 0.5μL 100x BSA
- 1.0μL KpnI-HF
- 1μL SphI-HF
- 50μL Total
- Incubate in 37°C water bath for 2 hours
Evening lab work done by: James Mathew, Maneesh Gupta, Claire Paduano
- Gel purified digested Avi-Tagged Backbone
- Ligation
