Wednesday, August 26, 2015

8 26 2015 28s CTM qPCR 1

Today I ran two reps of the 28s target, one on the opticon and one at the CFX. We may use this as a normalizing gene if the reads look consistent. This is the run on the opticon. 

Primers:
170228s_3_FWDTAAGGCCAGTGTGGGAGAGAJH8/12/2015205559.88O.lurida"28S ribosomal protein S5, mitochondrial (MRP-S5) (S5mt)"Q5REJ1
170128s_3_REVCGCCTCACTTTTTGTGCCTCJH8/12/2015205560.04O.lurida"28S ribosomal protein S5, mitochondrial (MRP-S5) (S5mt)"Q5REJ1


Reagent Table:
    VolumeReactions X80
    Ssofast Evagreen MM 10800
    FWD Primer0.540
    REV Primer0.540
    PCR H2O5400
    1:9 cDNA4
  1. Added reagents from greatest to least volume
  2. Vortexed
  3. Centrifuged briefly
  4. Pipetted 11 ul Master Mix to each tube using a  multichannel pipetter
  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:
12345678910
DNased 42215 HC1DNased 42215 NC1DNased 42215 SC1DNased 42215 HT1 1DNased 42215 NT1 1DNased 42215 ST1 1DNased 42215 HM1 1DNased 42215 NM1 1DNased 42215 SM1 1NTC
DNased 42215 HC2DNased 42215 NC2DNased 42215 SC2DNased 42215 HT1 2DNased 42215 NT1 2DNased 42215 ST1 2DNased 42215 HM1 2DNased 42215 NM1 2DNased 42215 SM1 2NTC
DNased 42215 HC3DNased 42215 NC3DNased 42215 SC3DNased 42215 HT1 3DNased 42215 NT1 3DNased 42215 ST1 3DNased 42215 HM1 3DNased 42215 NM1 3DNased 42215 SM1 3NTC
DNased 42215 HC4DNased 42215 NC4DNased 42215 SC4DNased 42215 HT1 4DNased 42215 NT1 4DNased 42215 ST1 4DNased 42215 HM1 4DNased 42215 NM1 4DNased 42215 SM1 4NTC
DNased 42215 HC5DNased 42215 NC5DNased 42215 SC5DNased 42215 HT1 5DNased 42215 NT1 5DNased 42215 ST1 5DNased 42215 HM1 5DNased 42215 NM1 5DNased 42215 SM1 5
DNased 42215 HC6DNased 42215 NC6DNased 42215 SC6DNased 42215 HT1 6DNased 42215 NT1 6DNased 42215 ST1 6DNased 42215 HM1 6DNased 42215 NM1 6DNased 42215 SM1 6
DNased 42215 HC7DNased 42215 NC7DNased 42215 SC7DNased 42215 HT1 7DNased 42215 NT1 7DNased 42215 ST1 7DNased 42215 HM1 7DNased 42215 NM1 7DNased 42215 SM1 7
DNased 42215 HC8DNased 42215 NC8DNased 42215 SC8DNased 42215 HT1 8DNased 42215 NT1 8DNased 42215 ST1 8DNased 42215 HM1 8DNased 42215 NM1 8DNased 42215 SM1 8
All 
Amplification
Melt Curve
NTCs
Amplification
Melt Curve

The amplification and melt curves look good. There's some minor amplification in the NTCs but it appears to be a random product larger than the target so there's no worry about it. 

You can find the raw ct value data here and the raw data here.

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