|2x Apex Red||12.5||125|
|Forward Primer (10uM)||0.5||5|
|Reverse Primer (10uM)||0.5||5|
Using the final concentration I mixed each master mix going from largest to smallest volume.
I then pipetted 24 ul in each well followed by 1 ul of water or sample depending.
Then I ran the following PCR program:
|95 C||5 min|
|95 C||30 sec|
|55 C||30 sec|
|72 C||30 sec|
|repeat steps 2-4 40 times|
|72 C||3 min|
Once the PCR finished I ran 25 ul of each product on a 1.3% agarose gel
Gel Reagent Table
|1X Low TAE||175 ml|
- Add agarose to TAE.
- Microwave 1 minute stir
- Repeat until no particulate matter in solution
- Add EtBr while agarose still hot
- Gently pour in one corner of the gel cast until tray is full
I then ran the gel at 100 v for 40 minutes. I placed it on the transilluminator to view any bands that may have formed. In my haste I forgot to image the gel, Luckily the gel ran fine with strong banding and minor streaking due to over filled wells.
After running the gel I excised the bands. Once the bands were cut out of the gel and placed in a Millipore Purification Column and centrifuged at 5,000 rcf for 10 minutes to collect the purified DNA.
To send them off for sequencing I plated 4 replicates of 10 ul for each sample in the appropriate tube. On a second plate I plated two replicates of 3 uM primer working stock for each appropriate sample. These plates are now being held in the 4 C in 213 for sequencing.