DNA extraction from Gel Slices and the 2nd PCR Reaction

The DNA bands in the gel will be extracted using a QIAquick gel extraction kit, and then a 2nd PCR reaction will be done using the purified DNA as teh template.

QIAquick Gel Extraction Protocol

1. Weight the gel slice in a tube, making note to subtract the weight of the tube itself.
2. Add 3 volumes of Buffer QG to 1 volume of gel. For this experiment it will be 630 microliters and 480 microliters for the A and B gels respectively.
3. Incubate at 50 degrees Celsiu for 15 minutes.
4. After the gel discolces completely, (noting that the mixture is a yellow colour), add 1 gel volume of isopropanol to each sample and mix.
5. Place the QIAquick spin colum in a 2ml collection tube and put sample in.
6. Centrifuge for 1 minute to bind the DNA
7. Discard flow-through and place QIAquick column back in teh same collection tube.
8. Add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1 minute.
9. Discard the flow-through and centrifuge thet QIAquick column for an additional minute, at 13,000 rpm.
10. Place the QIAquick column into a clean 1.5 ml microcentrifuge tube.
11. Elute the DNA by adding 40 microliters of elution buffer to the center of the QIAquick column membrane. allow it to stand for 1 minute, then centrifuge for 1 minute.

2nd PCR Reaction Protocol

Into a single eppendorf tube, add via pipet:

1. 40.5 microliters of H2O
2. 1 microliter of Primer 1 ((the forward start primer)
3. 1 microliter of Primer 2 (the reverse primer with teh stop codon)
4. 1 microliter of dNTPs
5. 1 microliter of the 10X PCR Buffer
6. 1 microliter of Vent Polymerase
7. 1 microliter of the template DNA from PCR A
8. 1 microliter of the template DNA from PCR B
9. Mix by pipeting 40 microliter volumes 2-3 times.

The toal volume should now be 51 microliters. Place this mixture into the PCR machine, set at 95 degrees Celsius. Then have one cycle of 5 minutes of denaturation at 95 degrees Celsius. Then have 30 repitions of this set:

1. Denaturation at 95 degrees Celsius for 1 minute
2. Annealing at 50 degress Celsisus for 1 minute
3. Extension at 72 degress Celsisus for 1 minute

Followed by a single 10 minute extension at 72 degress Celsisus.