Tuesday, May 26, 2015

5 26 2015 Oly cDNA/Primer Check

Today I ran a qPCR using the Actin, HSP70, and Glutamine synthetase primers used by the 310 class on 6 cDNA samples prepared by Sam last week. You can read about it here. I made fresh working stocks from the original primer stocks. As per Sam's suggestion I diluted the cDNA to a 1:4 dilution to save on cDNA so I can run multiple primer checks.

Primer Working Stocks:

Actin
Ol_Act_FGACCAGCCAAATCCAGACGABC6/13/2012205560O.luridaActin, adductor muscle
Ol_Act_RCGGTCGTACCACTGGTATCG6/13/2012206060O.luridaActin, adductor muscle 

HSP70

HSP70_gigas_FGTTCCGATTTGTTCCGTGCCKK20C.gigasHSP70
HSP70_gigas_RTTGTCGCCATTTTCCTCGCTKK20C.gigasHSP70
Glutamine synthetase

Gluta_FATTCCCTCCCCTGATCCGTCCACCCAO.luridaGlutamine synthetase
Gluta_RTGGGTGGACGGATCAGGGGAGGGAATO.luridaGlutamine synthetase
Working Stock Calculations:
ul
Primer10
Nuclease free H2090
cDNA Dilution Calculations:
cDNA 1:4 Dilution
cDNA 10
Nuclease free H2030
Final Volume40
I then made up enough master mix to run 22 reactions. I miscalculated the number of no template controls (NTCs) I needed. I made too much master mix  for this run and will cut back on the next run.  
Master Mix Reagent Table:
VolumeReactions X12
Ssofast Evagreen MM 10220
FWD Primer0.511
REV Primer0.511
Nuclease Free H2O8176
cDNA1
To make each master mix, 3 total with one mix for each set of primers, I added the volumes from greatest to least of the reagents. 

Then I pipetted the master mixes into the plate followed by 1 ul of either water, positive control, or sample. I made duplicates of each sample to ensure the results. The positive control was from the NF31 Seed oyster DNA Isolation from 3/23/2015 with a concentration of 117.53 ng/ul. 

Plate Layout:
ActinActinHSP70HSP70GlutaGluta
789101112
C+NTCC+NTCC+NTC
NT1 NT1 NT1NT1NT1NT1
HT1HT1HT1HT1HT1HT1
ST1ST1ST1ST1ST1ST1
NC1NC1NC1NC1NC1NC1
HC1HC1HC1HC1HC1HC1
SC1SC1SC1SC1SC1SC1
NTCNTCNTCNTCNTCNTC
The results from the qPCR are below:
Actin Amp

Actin Melt Curve

HSP70 Amp

HSP70 Melt Curve

Glutamine Synthetase Amp

Glutamine Synthetase Melt Curve

Something was wrong with the Positive control which failed to amplify with two of the three primers. It also appears that glutamine synthetase indescriminately amplifies as it amplified in all three of the NTCs. Finally it appears that only 1 of the HSP samples amplified early than the controls. 

I can rerun this qPCR tomorrow with a better positive control but I believe the Actin and HSP70 primers still work well while the glutamine synthetase primer does not. I will begin running the new primers when they come in. 

You can see the raw qPCR data here.


2 comments:

  1. Actually I think there is an issue with all reactions, with regard to # of products. A "positive" control is not needed. As per GS- it is likely contamination versus "indescriminately amplifies". There should also be an analysis of the duplicates to see consistency.

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