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David Roche 11:53, 19 August 2009 (EDT)

PAH Site-Directed Mutagenesis

There are 2 sites in PAH that require site directed mutagenesis (one XbaI site and one PstI site). We chose to start with the XbaI site, as this would mean we could start to BioBrick the gene if successful.

The PAH gene is 1359bp long. It is contained in the vector pDNR-LIB which is 4kb long.
The primers used were PAH t201a fwd and rev.

The mastermix composition was as follows:
125ul total
92.5ul H20
5ul DNA (PAH midi prep) (10ng/ul concentration - this required us diluting our 4500ng/ul solution with ratio 1:450)
2.5ul dNTPs
12.5ul Pfu Ultra Buffer
2.5ul Pfu Ultra

23ul for each sample. The positive samples should have 1ul of both the forward and reverse primers. The negative control should have 2ul of water added.

PCR Protocol

Ideal PCR temps :
Fwd - 54.3 w/out, 66.0 with
Rev - 54.0 w/out, 64.0 with

1 cycle : 30s at 95'
16 cycles : 30s at 95'; 45s at 50', 52', 54'; 3m at 68'

Annealing temperatures of 50, 52, 54 degrees were used, and elongation of 68 degrees. Negative Control was performed at 50 degrees.

After PCR the products were run on a gel.
Lane 1: Ladder; 2 = 50'; 3 = 52'; 4 = 54'; 5 = negative

Results of Gel

The gel showed a normal ladder. the 3 positive samples showed no distinct bands, but a band for the primers. The negative contol showed no bands.
This is possibly due to the dilution factor used for the DNA midi sample (1ul into 450ul H20, hard to accurately pipette 1ul).

Will try again tomorrow using a 2ul in 900ul dilution for the DNA. This should give a more accurate concentration.

Gel Purification : OtsA and Dam

Royah, David and Dineka
After OtsA and Dam were run on a gel, the bands of interest were cut out using a UV viewing plate and a scalpel. This gel was then put into an eppendorf, and gel extraction was run to purify the DNA out. About 30ul of DNA of each was purified

Biobricks extracted from the registry: