Tim: PCR of cat gene, February 10
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Contents |
Aim
The purpose of thi sexperiment (caught your attention didn't I?) was to amplify the cat gene for later use in transformant selection.
Methods
Hotstar Taq Protocol as by Farren Issacs's guide.
Reagent mixture: Image:Table1.jpg
The following PCR program was programmed into PCR machine 4 under the name Farren.
Heated Lid: Yes
Pause: No
(95 for 15min)
(94 for 30s --> 56 for 30s --> 72 for 1.5min)x7
(94 for 30s --> 65 for 30s --> 72 for 1.5min)x30
(72 for 5min --> 4 for infinite)
Preheat lid: Yes
Results
Image:Tim Schmidt's PCR Expt -1 Gel Picture with Ladder.JPG
1. Lane 5 represents the PCR product of a normal reaction mixture with template but without taq.
2. Lane 6 represents the PCR product of a normal reaction mixture with taq but without template.
3. Lane 7 represents the PCR product of a normal reaction mixture with taq and template.
4. Lanes 4 and 8 are 1 kb+ ladders and the rest of the lanes are water.
Discussion
The strong band in the template negative control (lane 6) was unexpected. This band could be due to template contamination of either that particular PCR preparation, or possibly a contamination of one of the reagents. Both bands (lanes 6,7) are approximately 1.1 kb long, which corresponds to the expected length of the cat gene.
