Thursday, June 25, 2015

6 25 2015 Flanking PCR pt 4

Yesterday I ran a PCR for some flanking primers that failed to get good coverage based on Steven's analysis. Today I ran the products on a gel and excised the bands for sanger sequencing.

1660Flk_GRB2_FWDTCAGAACTGGTTCAAAGCTGAGTJH6/1/20152360O.luridaGRB2 FlankingP62994
1658Flk_H3.3_FWDCCAATGACAAATGAGCCACACAAJH6/1/20152360O.luridaH3.3 FlankingQ6P823
1656Flk_H2A.V_FWDGCGATGGAGTTGATGAGGTGJH6/1/20152059O.luridaH2A.V FlankingP08991
1652Flk_p29ING_FWDGTGGACACACATGCACTCCTJH6/1/20152060O.luridap29ING4 FlankingQ8C0D7

Reagent Table:
Reaction_ComponentsVolumeFinal Concentration
2x Apex Red12.5125
Forward Primer (10uM)0.55
Reverse Primer (10uM)0.55

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 C5 min
95 C30 sec
55 C30 sec
72 C30 sec
repeat steps 2-4 40 times
72 C3 min
4 CHold
Once the PCR finished I ran 25 ul of each product on a 1.3% agarose gel

Gel Reagent Table
1X Low TAE175 ml
Agarose2.3 g
EtBr17.5 ul

  1. Add agarose to TAE.
  2. Microwave 1 minute stir
  3. Repeat until no particulate matter in solution
  4. Add EtBr while agarose still hot
  5. 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. 

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