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.
- 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.
