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From 2010.igem.org

Contents 1 Nina 1.1 Gel verification of protein A (ZZ domain) and IgG protease 1.2 Colony-PCR of site-directed mutagenesis Tyrosinase 1.3 Overnight culture of protein A (ZZ domain) and Tyrosinase 1.4 Sequencing of protein A (ZZ domain)

Nina

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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:

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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:

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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.

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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