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Team:Cornell/Week 8
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Revision as of 02:22, 29 July 2011 by Rockyshell92 (Talk | contribs)
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July 24th - July 30th
Sunday, July 24
Monday, July 25
Tuesday, July 26
Lab work done by: Claire Paduano & ?
Lab work done by: Charlie Chung, Nicholas Kramer & Jim Mathew
- Objectives
- To anneal primers containing the Avi-Tag 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 + Avi-Tag + 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 + Avi-Tag + 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 + Avi-Tag + 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
- Avi-Tag + 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 waterbath 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 DH5-alpha electrocompetent cells with ampicillin resistance.
- Samples
- Avitag + pZE12 (backbone)
- VoiE + Avitag + pZE12
- GFP + Avitag + PZE12
- RFP + Avitag + pZE12
- GFP + pZE12
- pZE12 control
- VioE control
- Avitag control
- Samples
Thursday, July 28
Lab work done by Youjin Cho, Bill Jo, and Nancy Li
- Mini-prepped results of transformation of ligation products. Sending in for sequencing now.
Sequencing Order #: 10253750
