IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/06/21
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Today, the objective is to obtain PCR products of Arabadopsis allergens LTP 1, Ger 3, and Bet v1.
1. Obtain wildtype Arabadopsis genomic DNA 2. PCR for each LTP 1, Ger 3, and Bet v1
1. Obtain wildtype Arabadopsis genomic DNA
Obtained from the Harvard Herbaria. Nanodrop yields 15.7 ng/μL.
2. PCR for each LTP 1, Ger 3, and Bet v1
Only LTP sense, Ger 3 sense, and Ger 3 antisense were successful. Concentrations were 2.6, 7.8, and 7.9 ng/μL, respectively.
The other genes were not amplified. There are many reasons for why LTP antisense and Bet v1 did not amplify, so we tried to eliminate some potential problems. After checking the primers against the arabadopsis genome, the primers seem correct. Annealing temperatures also fall within the appropriate range.
Other possible problems could be contamination, improper pH, insufficient DNA, primer design beyond sequencing (unlikely), or incorrect primers in a reaction (unlikely). We will obtain more arabadopsis genomic DNA and try PCR again.
Barnase and Barstar
Barnase and Barstar Plasmids from ADDGENE arrived in mail
pMT316 - Barstar. Info from ADDGENE
pMT413 - Barnase, Barstar. Info from ADDGENE
pMT1002 - Barnase, Barstar. Info from ADDGENE
Notes from lab meeting
Karmella suggested we try adding an LVA tail to the Cre we use for the crelox fence system.
GAL4 DBD Innoculation
Pipetted 5mL of LB+Amp each into capped cc tubes, and mixed bacterial scrapings from 5 separate colonies of transformed GAL4 DNA binding domain E.Coli. Set to shake at 37°C overnight.
LacI (O2, E10) PCR, take 2
repeating PCR on O2 and E10 after first attempt failed.
20μL in each PCR tube
.5μL polymerase 1μL LacIn.BB.Rev 1μL LacIn.BB.Fwd 2μL DNTP 4μL 5x buffer, vortexed 1μL sample 10.5μL H2O
Used modified LACIN PCR program:
1 = 95°C for 10:00 mins
2 = 95°C for :15 seconds
3 = 50°C for :30 seconds
4 = 72°C for 1:30 mins
5 = goto 2, 29 times
6 = 72°C for 10:00 mins
7 = 4°C forever
Cre, Lox66, Lox 71, GFP, and Luciferase Bacterial Transformation
Performed bacterial transformation according to Silver:_Bacterial_Transformation, however we used 1μL in step 3, and used entire tube of TOP10 competent cells instead of only 10-15μL.
Cre DNA Recombinase
GFP mutant (phenotype identical to wildtype)
Labeled each plate LB+Amp, its contents (ie Lox71 or Firefly Luciferase), MP, JW, OM, 6-21-10
Set all five plates to incubate at 37°C overnight.