Overlap PCR

 

We started this week off with a get extraction. We first measured the peices of gel, combined it with four volumes of buffer, and nanodropped the readings. 

Tet gel sample volume - 248.3 mg, 993.2 μl buffer, and nanodropped at 21.6 

Left  gel sample volume - 192.9 mg, 771.6 μl buffer, and nanodropped at 10.1


We then used those two samples in overlap PCR, doing a heat gradient to help find our best annealing temperature. We diluted the tet sample with 16 μl tet and 48 μl PCR water. As we didn't get enough left sample from the gel, we also diluted an old left sample with 15 μl of left and 30 μl of PCR water. We also used an old right sample as we didn't get any form the gel extraction, and luckily enough we didn't need to dilute that. 

We had eight tubes of left and tet, and one tube of left, tet and right just to try. 


1 LT 54°C 

2 LT 52.3°C 

3 LT 49.7°C 

4 LT, LTR 46.3°C 

5 LT 41.7°C 

6 LT 37.7°C 

7 LT 35.3°C 

8 LT 34°C        


After running PCR and amplifying, we ran the samples on a gel. 



While the 34°C and 54°C  samples are clear, the ones between them are very faint. the LTR one is a streak as expected, but the LT samples should have been clearer. Next week we will run the entirety of the samples to se as much DNA as possible. 



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