Wednesday, June 10, 2015

6 10 2015 Flanking Primer PCR pt. 2

Yesterday I ran a PCR using a couple flanking primers I created to see if they worked. The primers worked successfully, which you can read about here. Before the end of the day I ran the remaining 11 primers using the same protocol. Today I ran gels on all of the products to see which ones worked and isolate the PCR product for sequencing.

Primers

1672Flk_PGRP_FWDAGCTGGTGCAGTCCTATCAGJH6/1/20152059O.luridaPGRP-S FlankingO75594
1671Flk_PGRP_REVTGTGTATGAAAAGTAATGAAGAGCAJH6/1/20152557O.luridaO75594
1670Flk_CARM_FWDTTCACACAGCCCATTGTGGATJH6/1/20152160O.luridaCARM1 FlankingQ6DC04
1669Flk_CARM_REVTGGGATGGGTCAGATAAACCTJH6/1/20152158O.luridaQ6DC04
1668Flk_BMP2_FWDGGCTGGCTGGATCGTCATJH6/1/20151860O.luridaBMP2 FlankingP12643
1667Flk_BMP2_REVATGGAGTCTGTGGACGGTTTGJH6/1/20152160O.luridaP12643
1666Flk_HSPb11_FWDAGAATTGTCTGTGGAATCGAGCJH6/1/20152259O.luridaHSPb11 FlankingQ9Y547
1665Flk_HSPb11_REVATCAACGCCAGGGGAACTTGJH6/1/20152061O.luridaQ9Y547
1664Flk_PGE/EP4_FWDGCTCAACGAATTGCTCTACTCCJH6/1/20152259O.luridaPGE/EP4 FlankingP32240
1663Flk_PGE/EP4_REVTCCGTCTGCTTTTTAGAATGGTAJH6/1/20152358O.luridaP32240
1662Flk_GABABR_FWDGAGGAGGACACGAAACTCCGJH6/1/20152060O.luridaGABABR FlankingQ9WV18
1661Flk_GABABR_REVTGCACCACACTCCTGATGACJH6/1/20152060O.luridaQ9WV18
1660Flk_GRB2_FWDTCAGAACTGGTTCAAAGCTGAGTJH6/1/20152360O.luridaGRB2 FlankingP62994
1659Flk_GRB2_REVACTGCGCTGACATACTGGACJH6/1/20152060O.luridaP62994
1658Flk_H3.3_FWDCCAATGACAAATGAGCCACACAAJH6/1/20152360O.luridaH3.3 FlankingQ6P823
1657Flk_H3.3_REVTCGTACAAAGCAAACTGCACGJH6/1/20152160O.luridaQ6P823
1656Flk_H2A.V_FWDGCGATGGAGTTGATGAGGTGJH6/1/20152059O.luridaH2A.V FlankingP08991
1655Flk_H2A.V_REVCAAGGCAGTTTCTCGTTCGGJH6/1/20152059O.luridaP08991
1654Flk_H2A_FWDTGCTGGGGTTTTTCTGGGTCJH6/1/20152060O.luridaH2A FlankingP02270
1653Flk_H2A_REVTCAGGACGTGGTAAAGGAGGAJH6/1/20152160O.luridaP02270
1652Flk_p29ING_FWDGTGGACACACATGCACTCCTJH6/1/20152060O.luridap29ING4 FlankingQ8C0D7
1651Flk_p29ING_REVAAGCAGACTCAGATTCAGGCJH6/1/20152058O.luridaQ8C0D7

Using the Apex Red PCR Master Mix I created master mixes for each set of primers and ran them together. 

Reagent Table

Reaction_ComponentsVolume (ul)Final Concentration
2x Apex Red12.5125
Forward Primer (10uM)0.55
Reverse Primer (10uM)0.55
H2010.5105
1:2 cDNA1
25
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:

TempTime
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 the products on a 1.3% agarose gel

Gel Reagent Table
ReagentVolume
1X Low TAE175-200 ml
Agarose2.3-2.6 g
EtBr17.5-20 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 gels at 100 v for 35 minutes. I placed it on the transilluminator to view any bands that may have formed. The gels are below. 

Gel 1 layout:
PGRP-SCARMBMP2
LadderNT1HT1ST1NC1HC1SC1NTCLadderNT1HT1ST1NC1HC1SC1NTCLadderNT1HT1ST1
BMP2HSPb11PGE/EP4
NC1HC1SC1NTCLadderNT1HT1ST1NC1HC1SC1NTCLadderNT1HT1ST1NC1HC1SC1NTC


Gel 2 layout:
GABABRGRB2H3.3
LadderNT1HT1ST1NC1HC1SC1NTCLadderNT1HT1ST1NC1HC1SC1NTCLadderNT1HT1ST1
H3.3H2A.VH2A
NC1HC1SC1NTCLadderNT1HT1ST1NC1HC1SC1NTCLadderNT1HT1ST1NC1HC1SC1NTC
p291N4
LadderNT1HT1ST1NC1HC1SC1NTCLadderEmptyEmptyEmptyEmptyEmptyEmptyEmptyEmptyEmptyEmptyEmpty


In Gel 1, the PGRP-S flanking primer failed completely. Also the Oyster Bay heat treated samples failed to amplify with the CARM and PGE/EP4 flanking primers. These did amplify previously so I'm not sure what it means. 

In Gel 2, The Heat treated Dabob sample failed to amplify with the GABABR primer again not sure what it means. Interesting, the Fidalgo heat treated sample's PCR product in the H3.3 region has a much higher molecular weight. Steven suggested this could be an alternative splicing which would be interesting. 

All bands except those in the failed PGRP primer were cut out and placed in either labelled 1.5 ml tubes or labelled Millipore Purification columns. The ones in the column were centrifuged for 10 minutes at 5000 rcf. The purified solutions and gel bits are stored in the 4 C fridge in 209. When we get more purification columns either I or Sam will spin down the remaining bands. Once the samples are isolated they will be sent off for sequencing. Hopefully the sequences will be the same in all populations so that the qPCR's can be trusted. That or they are so wildly different they expose significant differences in the genes between populations. Either way it should be interesting. 
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