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:

	Volume	Reactions X24
Ssofast Evagreen MM	10	240
FWD Actin Primer	0.5	12
REV Actin Primer	0.5	12
Nuclease Free H2O	8.5	204
cDNA	0.5

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:

9	10	11	12
C-	C+	C-	C+
42715ST1	42715HT1	42715SM1	42715HM1
42715ST2	42715HT2	42715SM2	42715HM2
42715ST3	42715HT3	42715SM3	42715HM3
42715ST4	42715HT4	42715SM4	42715HM4
42715ST5	42715HT5	42715SM5	42715HM5

I ran the following program:

Sybr New Plate+Sybr cDNA 55 melt 2
Step	Temperature	Time
Initiation	95 C	10 min
Elongation	95 C	15 sec
	55 C	15 sec
Read
	72 C	15 sec
Read
Repeat Elongation 40 times
Termination	95 C	1 min
	55 C	1 sec
Melt Curve Manual ramp 0.2C per sec Read 0.5 C	65 - 95 C	30 sec
	21 C	10 min
End

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.