Team:Stockholm/13 August 2010

From 2010.igem.org

Nina

Gel verification of protein A (ZZ domain) and IgG protease

I ran a new agarose gel 1 % on my protein A and IgG protease samples. However I didn't load the positive protein A control sample since I realized that it would not fullfill its purpose considering the vector verification primer I used does not bind to the vector protein A sits in, which is the original vector I obtained it in.

Ladder: GeneRuler™ DNA Ladder Mix, ready-to-use, 100-10,000 bp Fermentas

Arrangement on gels:

Colony-PCR of site-directed mutagenesis Tyrosinase

Colony nr: 2, 4, 6 & 8.

PCR Mix:

Total volume 100 ul, aliquate 25 ul in the four pcr tubes.

F primer 50 uM 1 ul
R primer 50 uM 1 ul
dNTP 10 uM 1 ul
Buffer phusion 5X 20 ul
Phusion polymerase 1 ul
H2O 75 ul

PCR prgm:

98 °C 2 min

22 cycles of:

98 °C 30 sec
50 °C 30 sec
72 °C 2 min

72 °C 1 min

4 °C ∞

I ran 5 ul of the PCR products with 1 ul loading dye 6X on an agarose gel 1 % 80 V.

Ladder: GeneRuler™ DNA Ladder Mix, ready-to-use, 100-10,000 bp Fermentas

Arrangement on gels:

Overnight culture of protein A (ZZ domain) and Tyrosinase

I inoculated protein A ZZ domain inserted in the bank vector C (with chloramphenicol) colony # 5 in 12 ml LB and 24 ul chloramphenicol 50 mg/ml. This was incubated O/N in 37 °C with shake.

I inoculated tyrosinase in its original vector colonies # 2, 4, 6 & 8 in 12 ml LB and 24 ul ampicillin 50 mg/ml. This was incubated O/N in 37 °C with shake.

Sequencing of protein A (ZZ domain)

I prepared a tube of protein A ZZ domain for sequencing:

15 ul plasmid from a mini-prep and 1.5 ul (10uM) primer. Preferably the forward primer (VF) of the vectors verification primers.

ASB0045 303

Andreas

Site-directed mutagenesis of SOD & yCCS

Transformation results

SOD-->Amp 100: Few colonies, probably due to contaminated competent cells.
SOD-->Cm 25: Good colony yield
yCCS-->Cm 25: Good colony yield

Colony PCR

SOD-->Amp: 4 colonies (SA1-SA4)
SOD-->Cm: 5 colonies (SC1-SC5)
yCCS-->Cm: 5 colonies (yC1-yC5)
Positive control: pSB1C3.BBa_J18932 plasmid (PC)

Procedures as described in the colony PCR protocol. Elongation time: 1:40

yCCS digestion

Amplified yCCS DNA was digested to verify successful site-directed mutagenesis (i.e. EcoRI site removal).

2 μl 10X FD Green buffer
17 μl DNA
1 μl FastDigest EcoRI

Incubation: 37 °C, 10 min

Gel verification

Colony PCR verification of mutagenized yCCS and SOD.
1 % agarose, 110 V, 40 min.
1st row:1 kb λ, SA1, SA2, SA3, SA4, SC1, SC2, SC3, SC4, SC5, d.yC5
2nd row: 1 kb λ, yC1, d.yC1, yC2, d.yC2, yC3, d.yC3, yC4, d.yC4, yC5, PC
λ: DNA ladder. d.:digested sample.

1 % agarose, 110 V, 40 min