IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/06/21

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Procedures
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

Results
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.

Transformed the following each into its own tube of TOP10 E.Coli:

Cre DNA Recombinase
 * biobrick BBa_J61047, located on plate 1, well 5D, plasmid pSB1A2

Lox71
 * biobrick BBa_I718017, located on plate 1, well 17J, plasmid pSB1A2

Firefly Luciferase
 * biobrick BBa_I712019, located on plate 1, well 10H, plasmid pSB1AK8.

GFP mutant (phenotype identical to wildtype)
 * biobrick BBa_E0040, located on plate 1, well 14K, plasmid pSB1A2.

Lox66
 * biobrick BBa_I718016, located on plate 1, well 17H, plasmid pSB1A2.

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.