Lab entry 12

After double digesting and ligating an adapter to the DNA, we did a test PCR. We did this to test for successful library construction of the samples.

  1. First, we made a master mix for the RADSeq using the following:
Master Mix Rxn: 1 Rxns: 11
NEB One-Taq 2x Master Mix 8 uL 88 uL
Forward Primer (10mM) PCR1X .4 uL 4.4 uL
Reverse Primer (10mM) PCR26 .4 uL 4.4 uL
Pure H2O 6.2 uL 68.2 uL
Master Mix Total: 15 ul 165 ul
Library DNA Template 1 ul  
Total reaction volume 16 uL  


2. We ran PCR1 on BIORAD #1/2 using 5 uL of the total reaction volume and 2 uL of loading dye.

3. Then, we ran the the products of PCR1 for each sample on 1.5% agarose gel with a 100 bp ladder at 130 V for 40 minutes.

The test PCR had samples #17-24.

For the final PCR, we used the same steps with the following master mix:

Master Mix Rxn: 1 Rxns: 11
Phusion DNA Polymerase .31 uL 3.41 uL
5X Phusion HF buffer 6.25 uL 69 uL
Forward Primer (10mM) PCR1_X 1.56 uL 17.2 uL
Reverse Primer (10mM) PCR2_1 1.56 uL 17.2 uL
DNTPs 10 mM .63 uL 6.93 uL
DMSO .94 uL 10.3 uL
Pure H2O 10.75 uL 118.25 uL
Master Mix Total: 22 ul 242.04 ul
Library DNA Template 3 ul  
Total reaction volume 25 uL  

We used the same samples #17-24. However, for samples 21 and 22 we used PCR27 as the reverse primer instead. This was a mistake made because we ran out of our original master mix and used another groups.



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