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From 2010.igem.org

Contents 1 Nina 1.1 Tranformation of protein A 1.2 Sequencing of tyrosinase 1.3 PCR on N_CPP cluster 2 Andreas

Nina

Tranformation of protein A

I transformed 100 ul BL21 cells with both 1 and 3 ul of protein A inserted into the peX vector. This transformation is carried out in order to perform an IPTG induction on protein A in BL21 e.coli cells.

The transformation procedure is described in protocols. However, in step 1 I thawed the cells in 15 min instead of 10 min. In step 2 I added 1 ul of DNA sample to 100 ul BL21 cells and 3 ul of DNA sample to an additional 100 ul BL21 cells.

Sequencing of tyrosinase

Since the last sequencing of the two tyrosinase samples did not turn out well, I send two new tyrosinase samples for sequencing. This time I mixed the sample with primers complementary to the bank vector iGEM send the gene in. I therefore mixed with the primer VR. I choose VR instead of VF2 because I wanted the primer to bind closer to the site where I performed a site directed mutagenesis.

I prepared two tubes of tyrosinase:

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

• Colony #4: ASB0045 B01

• Colony #6: ASB0045 B02

PCR on N_CPP cluster

We obtained our N_CPP in a lysophilized form in an eppendorf tube, to which I added 24 ul of dH2O, vortexed and spann down by centrifuging ~10 seconds.

I prepared a PCR mix with the N_CPP cluster as the DNA template. This PCR product will in following days become digested and ligated into both the pEX and shipping vector.

PCR mix:

1. Buffer Pfu buffer + MgSO4 10X 4.33 ul
2. dNTP 10 uM 1 ul
3. primer VF2 10 uM 3 ul
4. primer pgex 10 uM 3 ul
5. polymerase Pfu 1 ul
6. DNA template 1 ul
7. H2O 30 ul

I did a 1:5 dilution of primer pgex from 50 uM to 10 uM. 1 ul 50 uM primer and 4 ul dH2O.

PCR prgm:

Andreas