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 Test

Ran this WS cPCR using GoTaq and used a different aliquot of the RA primers working stocks.

Master mix calcs are here.

Results:

Ladder is Hyperladder V (Bioline)

Another failure. And, another inconsistent run (i.e. duplicates aren’t the same). Regardless, band is too faint for using the 3e7 copy plasmid. Had been using RA primers that Lisa had from an old class. Maybe I should make new working stocks??

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