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From 2010.igem.org

Contents 1 Nina 1.1 Mini prep on Tyrosinase 1.2 Fusionprotein of IgG protease & protein A (ZZ domain) 1.3 Digestion of protein A ZZ domain in bank vector C 1.4 Digestion of IgG protease in bank vector C 1.5 Ligation of the digest samples 1.6 Transform the ligate samples 1.7 Glycerol stock on protein A (ZZ domain)

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:

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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. 1:

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

1. 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.

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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