Monday, May 11, 2015

5 7 2015 DNase treatment and qPCR quality check

Last week was really busy so I'm just now catching up on writing these blog posts. After reviewing the previous quality checks with Steven and Sam, it was decided that the samples should be DNased again and checked once more. The DNase treatment was much more rigorous to eliminate any excess gDNA. 

DNase Rigorous Protocol:

1. Added 5 ul  or 10 ul DNase 10X Buffer to each 50 ul or 100 ul of RNA respectively.
2. Added 1.5 ul Turbo DNase to each RNA.
3. Incubated at 37 C for 30 minutes
4. Added 1.5 ul Turbo DNase to each RNA
5. Incubated at 37 C for 30 minutes
4. Added 12 ul or 24 ul DNase inhibitor to each 58 ul or 113 ul sample respectively
5. Incubated at room temp for 5 minutes, flicking occasionally
6. Centrifuged at 10,000 g for 1.5 minutes
7. Decanted the top 50 ul  or 100 ul of supernatant to fresh tube

After completing the DNAse treatment the samples were then run on qPCR to check for remaining gDNA contamination. 

qPCR Reagent Table:
VolumeReactions X24
Ssofast Evagreen MM 10240
FWD Actin Primer0.512
REV Actin Primer0.512
Nuclease Free H2O8.5204
1. Added each from greatest volume to least to make the master mix. 
2. Pipetted 20 ul master mix into each well of a qPCR partial plate
3. Added 0.5 ul sample to each tube

C- was the template control. C+ was the Fidalgo seed oysters extracted on 3/23/2015 with a concentration of 167.3 ng/ul.

qPCR Plate Layout:

I ran the following program:

Sybr New Plate+Sybr cDNA 55 melt 2
Initiation95 C10 min
Elongation95 C15 sec
55 C15 sec
72 C15 sec
Repeat Elongation 40 times
Termination95 C1 min
55 C1 sec
Melt Curve Manual ramp 0.2C per sec Read 0.5 C65 - 95 C30 sec
21 C10 min
You can see the amplification curves below:

Log Amplification Curve:

Melt Curve:

There still as some amplification of gDNA but far fewer samples had them. Based on my talk with Steven we decided to move forward with the cDNA process on Friday. 

You can see the raw qPCR data here.

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