Since the water source tests from 20140109 showed that water source had no effect AND the SPUD assay only amplified in wells that showed WSN1 amplification, I decided to try a different polymerase that I know is functional and performs well. I used 2x Sso Fast EvaGreen (BioRad) from the Roberts Lab instead of 2x Immomix (Bioline) from the Friedman Lab. Since the Sso Fast EvaGreen already contains a fluorescent SYBR analog, I did NOT use the RLP probe that is normally used in the WSN1 qPCR.
For template DNA, I used the serial dilution set from 20140109 of undigested pCR2.1/WS-3 in Roberts Lab Nanopure water (from 7/11/2012). Additionally, I ran a no template control with water and a no template control with no template added at all.
Master mix calcs are here (20140113 – WS qPCR SPUD SsoFast Test). See the Results below for plate layout, cycling params, etc. Baseline threshold was set at 400 RFUs for both fluorophores. All samples were run in duplicate (except the empty no template control; only a single well was run).
qPCR Report (PDF): Sam_2014-01-13 10-21-02_CC009827.pdf
qPCR Data File (CFX): Sam_2014-01-13 10-21-02_CC009827.pcrd
The results are interesting. Most importantly, the SPUD assay amplifies in all samples, including the negative controls. But, there is WSN1 amplification in all of the negative controls! Additionally, all of the WSN1 samples also amplify and exhibit the proper spacing between Cq for each 10-fold dilution (3.32 Cqs) for almost all of the samples. However, the SPUD assay still shows an equidistant shift in Cq when there’s a shift in WSN1 Cq. Also, it might be worth noting that the amplification levels (i.e. the raw fluorescent units) are way, way, way higher than what is normally produced using Immomix and the WSN1 probe.
This is too crazy. Lisa is going to try to run her old Hematodinium curve with the SPUD assay to see what happens.
Lisa ran her samples with three different polymerases (2x BioRad Sso Fast Probe, 2x SensiMix, and 2x Immomix). Her curves all look perfect and the SPUD assay amplified equally in all samples, including the WSN1 negative controls. Interestingly, the samples run with BioRad showed a general shift of ~3 Cqs EARLIER than the corresponding samples using the other two polymerases. This effect is also seen in the SPUD assay which comes up at ~30 Cq in the BioRad samples, but comes up at ~33 Cq in the other two polymerases. However, the BioRad exhibits some strange amplification patterns at the lower end of the standard curve that is not present in with the other two polymerases.
Lisa used different SPUD assay conditions than what I’ve been using. She used 0.3uL of the 1:10,000,000 per reaction. I have been using 2uL of the 1:10,000 template per reaction. Is it possible that my reactions are suffering inhibition due to molecular crowding (i.e. so much DNA in the reaction that the DNA is inhibiting the reaction)? Will test this out. Additionally, will have Lisa try her hand at running the newest WS standard curves to see if she has any luck.
Lisa also used Sigma PCR H2O instead of the Nanopure H2O.
Lisa also used a reaction volume of 21uL. This is odd, because it does not result in a 1x final concentration of the polymerases (she used 12.5uL of 2x polymerase mix in each reaction).
Lisa used different cycling parameters for run, which was for the Hematodinium curves (95C -15s, 60C – 30s, 72C 30s).