Monday, July 6, 2015

7 6 2015 Actin qPCR rerun

After noting some significant discrepancies between replicates last week we decided to change the protocol to better help optimize the process and make the data more consistent between runs. Today I'm running two replicates of Actin but this post will only cover the first one.

Primers:
1505Ol_Act_FGACCAGCCAAATCCAGACGABC6/13/2012205560O.luridaActin, adductor muscle
1504Ol_Act_RCGGTCGTACCACTGGTATCG6/13/2012206060O.luridaActin, adductor muscle 

To help with optimization I made a 1:9 dilution of cDNA so that pipette error would not affect the concentrations as much as it has previously. 

Reagent Table:
VolumeReactions X58
Ssofast Evagreen MM 10580
FWD Primer0.529
REV Primer0.529
1:9 cDNA9
  1. Added reagents from greatest to least volume
  2. Vortexed
  3. Centrifuged briefly
  4. Pipetted 11 ul Master Mix to each tube
  5. Pipetted 9 ul of 1:9 cDNA each column using a channel pipetter
  6. Centrifuged plate at 2000 rpm for 1 minute
  7. Ran Program Below
Program:
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 4NTC
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:

All Samples
NTCs

The samples look good. The NTCs did have amplification but it appears that it was primer dimers and nothing else. I'm running another plate over night and I'll post about it tomorrow as well as how the two replicates match. 

You can see the raw data file here.

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