Monday, June 22, 2015

6 22 2015 CARM1 qPCR

Today I ran a qPCR for CARM1 using cDNA created by Sam last week. On friday Steven got the initial run of sequenced samples to check that all populations produced the same sequence. He selected CARM1 as being the most similar with the other primers having one issue or another. I put together a qPCR plate with all the cDNA samples to see how CARM1 differs between populations and treatment/control.

Primer Used:
1642CARM1_FWDTGGTTATCAACAGCCCCGACJH5/21/20152055O.luridaHistone-arginine methyltransferase CARM1 (EC 2.1.1.-) (EC 2.1.1.125) (Coactivator-associated arginine methyltransferase 1) (Protein arginine N-methyltransferase 4)Q6DC04
1641CARM1_REVGTTGTTGACCCCAGGAGGAGJH5/21/20152055O.luridaHistone-arginine methyltransferase CARM1 (EC 2.1.1.-) (EC 2.1.1.125) (Coactivator-associated arginine methyltransferase 1) (Protein arginine N-methyltransferase 4)Q6DC04

qPCR Master Mix Reagent Table:
VolumeReactions X55
Ssofast Evagreen MM 10550
FWD Primer0.527.5
REV Primer0.527.5
Nuclease Free H2O8440
cDNA1
  1. Added reagents from greatest to least volume
  2. Vortexed
  3. Centrifuged briefly
  4. Pipetted 19 ul Master Mix to each tube
  5. Pipetted appropriate cDNA sample to each tube
    1. Due to pipette error only 3 NTCs were run
  6. Centrifuged plate at 2000 rpm for 1 minute
  7. Ran Program Below
Sybr New Plate+Sybr cDNA 60 melt 2 Read
StepTemperatureTime
Initiation95 C10 min
Elongation95 C30 sec
60 C1 min
Read
72 C30 sec
Read
Repeat Elongation 39 times
Termination95 C1 min
55 C1 sec
Melt Curve Manual ramp 0.2C per sec Read 0.5 C55 - 95 C30 sec
21 C10 min
End
Plate Layout:
1234567
DNased 42215 HC1DNased 42215 NC1DNased 42215 SC1DNased 42215 HT1 1DNased 42215 NT1 1DNased 42215 ST1 1NTC
DNased 42215 HC2DNased 42215 NC2DNased 42215 SC2DNased 42215 HT1 2DNased 42215 NT1 2DNased 42215 ST1 2NTC
DNased 42215 HC3DNased 42215 NC3DNased 42215 SC3DNased 42215 HT1 3DNased 42215 NT1 3DNased 42215 ST1 3NTC
DNased 42215 HC4DNased 42215 NC4DNased 42215 SC4DNased 42215 HT1 4DNased 42215 NT1 4DNased 42215 ST1 4
DNased 42215 HC5DNased 42215 NC5DNased 42215 SC5DNased 42215 HT1 5DNased 42215 NT1 5DNased 42215 ST1 5
DNased 42215 HC6DNased 42215 NC6DNased 42215 SC6DNased 42215 HT1 6DNased 42215 NT1 6DNased 42215 ST1 6
DNased 42215 HC7DNased 42215 NC7DNased 42215 SC7DNased 42215 HT1 7DNased 42215 NT1 7DNased 42215 ST1 7
DNased 42215 HC8DNased 42215 NC8DNased 42215 SC8DNased 42215 HT1 8DNased 42215 NT1 8DNased 42215 ST1 8

Results:

HC1-8 Amplification


 HC1-8 Melt Curve
NC1-8 Amplification
 NC1-8 Melt Curve
SC1-8 Amplification
 SC1-8 Melt Curve
HT1-8 Amplification
 HT1-8 Melt Curve
NT1-8 Amplification
 NT1-8 Melt Curve
ST1-8 Amplification
 ST1-8 Melt Curve
NTC 1-3 Amplification
 NTC 1-3 Melt Curve

As you can see there is good amplification amoung all samples but without running the data through PCR Miner I can't distinguish a difference. Also there was some amplification in the NTC but it appears to be nonspecific with a much higher melting temp than the actual products. These high temp amplifiers don't appear in any of the other samples so I'm not worried about them as possible contamination. I've run a PCR on some more flanking primers to have them sequenced and am running a qPCR for Actin to see if we have similar results.

You can see the raw data file here.
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