Friday, May 1, 2015

5 1 2015 qPCR Quality Check

Today I ran a quality check on the samples that I isolated yesterday. Instead of creating cDNA before doing the qPCR I ran just the RNA with Actin primers to determine if there was any genomic contamination in the RNA samples. I diluted the RNA to levels similar to that which would have gone from the cDNA process to the qPCR process. This was to ensure that any genomic dna would be at levels similar to that from from the cDNA process and wouldn't over estimate the concentration of them in the sample.

The qPCR protocol was as follows

Master Mix
VolumeReactions X12
Ssofast Evagreen MM 10120
FWD Primer0.56
REV Primer0.56
Nuclease Free H2O9.8117.6

1. Added the reagents from highest volume to lowest to the master mix
2. Vortexed
3. pipetted into .5 ml qPCR tubes
4. Added 0.2 ul RNA to each tube from each isolation
5. Placed into the Opticon 2 qPCR machine
6. Ran the program "Sybr New Plate + Sybr cDNA 55 melt 2 reads"


As you can see, the samples began amplifying at 27-30 cycles which is indicative of a genomic DNA contamination. The negative control did not begin amplifying until 37 cycles. 

For future work, I need to use a DNase treatment to remove genomic DNA before I create cDNA. Next week I will isolate the 310 heat shock animals, DNase treat everything, and produce cDNA. 

1 comment:

  1. You should add the following to your notebook:
    - Link to qPCR data file
    - Plate layout with sample names and well locations
    - Cycling parameters (cycling program name is insufficient because programs frequently get changed to customize cycling times and then saved under the same program name).
    - Clarify if negative control is actually a negative control sample or a no template control.

    Not needed for this entry, but just a heads up. After DNasing your RNA, you'll check it again via qPCR to verify that DNasing worked. Remember to include a positive control sample on that qPCR plate. Otherwise, if nothing amplifies there's no way to know if that was due to the success of the DNase treatment or a failure of the qPCR reactions.