Team:Stockholm/14 August 2010

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Revision as of 18:33, 25 August 2010

Nina

Mini prep on Tyrosinase

I performed a mini prep on overnight cultures of inoculated site direct mutagenesis tyrosinase from colonies #: 2, 4, 6 & 8. The method was according to the procedure described under Protocols.

spectrophotometer:

Fusionprotein of IgG protease & protein A (ZZ domain)

Digestion of protein A ZZ domain in bank vector C

DNA vector 2 ul
H2O 14 ul
10X fast digest buffer 2 ul
Restriction enzyme SpeI 1 ul
Restriction enzyme AgeI 1 ul

Digestion of IgG protease in bank vector C

This construct has previously been cut with the restriction enzyme NgoMIV (9/8-2010)and undergone a gel clean up.

DNA vector 2 ul
H2O 15 ul
10X fast digest buffer 2 ul
Restriction enzyme SpeI 1 ul

Incubated both digest samples in 37 °C for 5 min.

Ligation of the digest samples

I made two versions of the ligation: # 1 & 2.

1:

IgG vector 1 ul
protein A gene 1 ul
quick ligase 1 ul
2X ligase buffer 2.5 ul

2:

IgG vector 1.5 ul
protein A gene 2 ul
quick ligase 1 ul
2X ligase buffer 3 ul

Incubated both ligate samples in RT for 15 min.

Transform the ligate samples

I transformed both of the ligated samples in each 100 ul Top 10 cells. The transformation method is according to the procedure decribed in protocols. However in the step 1 I thawed for 15 min instead of 10 min. In step 2 I added 3 ul of DNA, in step 6 I used LB instead of SOC and finally in step 10 I spread 100 ul of sample on a chloramphenicol plate.

Glycerol stock on protein A (ZZ domain)

I made a glycerol stock on protein A ZZ domain inserted in the bank vector with chloramphenicol.

Glycerol stock:

400 ul Glycerol
800 ul Bacterial overnight sample

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 ===Fusionprotein of IgG protease & protein A (ZZ domain)===
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 ===Digestion of protein A ZZ domain in bank vector C===