Team:Stockholm/14 July 2010

From 2010.igem.org

Johan

DNA electrophoresis

Lane 1: Fermentas FastRuler™ High Range DNA Ladder ([http://www.fermentas.com/templates/files/tiny_mce/coa_pdf/coa_sm1123.pdf link]), 3: bFGF vector before site-directed mutagenesis PCR and Dpn I treatment (low concentration; 10 ng/µl), 5: bFGF vector after site-directed mutagenesis PCR and Dpn I treatment.

The vector (bFGF-pGEX) shows the correct size of ~5,5k bp. Comparison between lane 3 & 5 also shows that the amplification in the PCR worked and that Dpn I treatment did not cause problems.

Transformation

PCR creates linear DNA, to make the vector circular it has to be transformed; the E. coli makes it circular.

Notes on protocol:

Step 1. Two tubes Bl21 tubes with competent cells were used, one for the linear mutagenesis DNA vector, and one for commercial plasmid as a positive control.

Step 2. 1 µl was used for both transformations.

Step 3. LB was used instead of SOC.

Step 9. No dilutions was made

Step 10. 50 µl was plated. Two plates was used for each reaction, one commercial carbenicillin plate and one of our ampicillin plate (to examine the state of our plates).

Colony PCR

A colony PCR was performed with tubes from Andreas.

(make names more clear..)

PC 30 A
PC 31 A
PC 32 A
PC 30 B
PC 31 B
PC 32 B
PA 30 A
PA 31 A
PA 32 A
PA 30 B
PA 31 B
PA 32 B
PK 30 A
PK 31 A
PK 32 A
PK 30 B
PK 31 B
PK 32 B
PA SOD A
PA SOD B
PC SOD A
PC SOD B
PK SOD A
PK SOD B
PC yCCS A
PC yCCS B

All reaction used VF & VR2 primers. The primers were diluted 15x to get a concentration of 10 µM.
- 33 µm H2O
- 2 µl f.primer 10 µM
- 2 µl r.primer 10 µM
- 1 µl dNTPs 10 mM
- 10 µl Pfu buffer 10x
- 1 µl PfuX7 polymerase
- 1 µl template DNA

PCR Reaction:

98 °C - 2 min
31 cycles of
1. 98 °C - 10 sec
2. 55 °C - 15 sec
3. 72 °C - 3 min
72 °C - 5 min
4 °C - ∞

Andreas

Clonings

Colony verifications

Continued from 13/7

Restreaks

White colonies for all streaked/picked constructs except:

pSB1K3.SOD (a)
pSB1C3.SOD (b)
pSB1K3.BBa_J18931 (a)
pSB1A3.BBa_J18931 (b)
pSB1C3.yCCS (a)

Gel verifications

pEX.X constructs picked and amplified 13/7 were separated on a 1 % gel. No good results, gel results discarded. New gel to be run tomorrow.

Top10 ON cultures

One of the tubes lost its cap during ON incubation, so new ON cultures (5 ml LB + 100 μg/ml Amp) were set.

Protein expression of BBa_J18930-32 from pEX

BL21 ON cultures of pEX.BBa_J18930-32 were used to inoculate cultures for IPTG induction:

80 ml LB
1 % glucose
100 μg/ml Amp
0.8 ml ON culture

Culture grown in 37°C, 250 rpm until an OD₆₀₀ of ≈0.5. However, experiment was cancelled due to lack of time.

New 3 ml LB + 1 % glucose + 100 μg/ml Amp cultures were inoculated with same clones as picked 13/7 (pEX.BBa_J18930-32 A & B). Cultures grown in 30°C ON for glycerol stock preparation.