PCR – Withering Syndrome cPCR LoD Tests

Ran PCR starting with highest copy number of WS p16RK7 plasmid curve (from 20120731) due to lack of amplification of the 300 copy plasmid seen on 20130423.

Master mix calcs are here.

All samples were run in duplicate.

Results:

Ladder is Hyperladder V (Bioline).

Lowest copy number visible is 3e4 copies (30,000 copies). Will run subsequent PCRs with copy numbers between 30,000 and 3,000 to identify lower limit of detection for this withering syndrome cPCR assay.

PCR – Withering Syndrome cPCR LoD Tests

Ran PCR to determine limit of detection using dilutions starting at 300 copies (the lowest detectable copy number shown on Nate’s poster).

Master mix calcs are here.

Results:

Ladder (Hyperladder V; Bioline) is loaded in furthest right lane. It is smeared due to a loading error by Sonja who loaded her sample in the same well as the ladder.

No amplification. Will repeat with higher copy number plasmid templates.

PCR – Withering Syndrome cPCR Limit of Detection Test

Ran PCR using newly purchased GoTaq Flexi. Master mix calcs are here.

Results:

NOTHING??!!! Didn’t even bother with labelling because it’s not necessary. However, from left to right, it is the p16RK7 RLP plasmid curve from 3e7 – 3 copies, each in duplicate and a pair of NTCs.

PCR – Withering Syndrome cPCR Assay Limit of Detection Troubleshooting

Ran PCRs using the 3e7 copies of the p16RK7 plasmid curve used in the qPCR WS validation with some different Taq polymerases to see if the polymerase is the source of the

Results:

All samples were run in duplicate, except the GoTaq 1 Green Buff, where I accidentally loaded the 3e7 template into three of the four wells.

PCR – Withering Syndrome cPCR Assay Limit of Detection Test

Repeated yesterday’s PCR, but utilized the p16RK7 plasmid curve (from 20120731) from 3e7 to 3e1. All samples run in duplicate.

Results:

Ladder is Hyperladder V (Bioline)

Only detectable amplification is in the highest concentration, 3e7 copies! On the positive side, at least the bands are neat and clean with no smearing. Holy crap, this

PCR – Withering Sydnrome cPCR Assay Limit of Detection Test

Ran cPCR, similar to that performed on 20130206, but with a lower set of dilutions, since the previous cPCR successfully detected down to 30 copies.

All samples run in duplicate.

Results:

Gel Loading (left to right):

Hyperladder V (Bioline)

30 copies

30 copies

15 copies

15 copies

10 copies

10 copies

5 copies

5 copies

3 copies

3 copies

NTC

A band is present in all samples, except the NTC. However, I’m frustrated by the appearance of the bands that these cPCRs are producing. I don’t like the smearing and looking at Nate’s poster outside of the lab, he has gel images with tight, very defined bands with no smearing. Will discuss with Lisa.

However, it’s interesting that the cPCR seems to detect all the way down to 3 copies! This suggests that the qPCR and cPCR don’t exhibit any difference in their limits of detection. I find this a bit odd, but will wait until I speak with Lisa to see what she says about Nate’s gel images.

UPDATE: Lisa indicated that the cPCRs are performed with different reagents/recipes than the qPCR! Will give those a shot to see if that improves the band quality.