Ran qPCRs testing the “old” primers/probe (IDT primers, Biosearch Technologies Probe) using ABI 2x qPCR master mix and Promega 2x qPCR master mix.
The curve used was the p16RK7 from 20120731. This was used because it worked perfectly on 20140418.
Also ran a qPCR with all ABI components along with the following set of samples that Lisa needed checked for positive/negative of withering syndrome:
2010 Water Filter DNAs (Site 8 extracted 20120228 and Site 3 extracted 20120229 by me):
- SNI Site 8 0M Rep. 1
- SNI Site 8 +100M Rep. 1
- SNI Site 8 -100M #1
- SNI Site 8 +500M #1
- SNI Site 8 -500M #1
- SNI Site 3 0M #1
- SNI Site 3 +100M #1
- SNI Site 3 -100M #1
- SNI Site 3 +500M #1
- SNI Site 3 -500M #1
Master mix calcs are here: 20140422 – qPCR p16RK7 Curve Old Probe
Plate layout, cycling params, etc. can be found in the qPCR Report in the Results below.
All samples were run in duplicate.
NOTE: Since this used a different polymerase (ABI) than what has been used in the past for this assay (Bioline), I did NOT adjust the baseline threshold. A new baseline threshold will have to be decided upon.
The qPCR run with the new probe/primer set worked wonderfully! The qPCRs with the old primer/probes failed miserably!
Here are the amplification curves and standard curve graph from the new primer/probe sets using the ABI master mix. The brown colored curves are the standard curve. The blue colored curves are some of Lisa’s samples.
Below are the other TWO curves (yep, both of them!) run with the old primer/probe set. Purple colored curves are Promega master mix, green colored curves are ABI master mix.