DNR Geoduck Project- Michelle McCartha

SAFS- Roberts Lab
McCartha
02/05/16 Running Vp standard curve
Created by Michelle McCartha
Goal: To test complete set of the standard curve and determine if the primers and probe and curve are ready for running samples.
Methods:
Aliquoting primers and probe
C1V1=C2V2
     (100µM)(x)=(10µM)(100µL)x=10µL of each primer 
     100µL aliquot-10µL primer=90µL of nuclease free water
Added water then Primer stock solution and pipetted up and down to mix.
Inverted tubes and finger vortexed then centrifuged.
 
Master mix prep
Took out samples and allowed to thaw while making master mix. 
To make the master mix, the following volumes were used…including water in the master mix.
 
Master Mix Solutions Standard volume (μL) Multiply By new volume * 10% pipette error add  pipette error Final volume to add (μL)
Master mix 25 50 1250 125 1375 1375
FWD Primer 1.5 50 75 7.5 82.5 82.5
Rev Primer 1.5 50 75 7.5 82.5 82.5
Probe 1 50 50 5 55 55
Water 17 50 850 85 935 935
 
Water was added to the mix in accordance with the new method protocol.
To make the master mix, a 15ml centrifuge tube was used. The IQ Powermix was added first, followed by the FWD/REV primers and probe and finally the molecular water. 
The mix was finger vortexed as well as pipetted up and down to allow homogeneity. 
 
Plate Set up 
Set up for the plate is as follows:
 
1 2 3 4 5 6 7 8 9 10 11
A NTC 1Vp1 1Vp1 1Vp1 2Vp1 2Vp1 2Vp1 3Vp1 3Vp1 3Vp1
B NTC 1Vp5 1Vp5 1Vp5 2Vp5 2Vp5 2Vp5 3Vp5 3Vp5 3Vp5
C NTC 1Vp10 1Vp10 1Vp10 2Vp10 2Vp10 2Vp10 3Vp10 3Vp10 3Vp10
D NTC 1Vp25 1Vp25 1Vp25 2Vp25 2Vp25 2Vp25 3Vp25 3Vp25 3Vp25
E TC 1Vp50 1Vp50 1Vp50 2Vp50 2Vp50 2Vp50 3Vp50 3Vp50 3Vp50
F
G
H
Sample reactions were made by adding 46μL of the master mix followed by 4μL of the template.  
NTC was made by adding 46μL of master mix followed by 4μL of water
TC was made using 25 18-day manilla clams digested in 1ml Prok solution as all samples were digested. 
TC reactions were made by adding 46μL of the master mic followed by 4μL of the template. 
When all the reactions were prepped, checked to make sure the caps were on securely and centrifuged for 1 min at 2000RPM.
qPCR parameters
Plate was run at the following parameters:
    • Incubate 95C for 2 minutes 30 seconds
    • Incubate 95C for 30 seconds
    • Incubate 60C for 50 seconds
    • Plate read
    • Go to step 2, 39 more times

Saved tad file as 20160205_150807

Results:

Manila clam standard curve data is in the attached excel file.
 
 
 
 
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