PCR – Test Universal Primers with Abalone DNA

Since I’ve had no success in amplifying any of the Ireland Clam RLO (S/6/14 #19) DNA, I’m testing all the universal primer sets I’ve previously tried on the Ireland Clam DNA with red abalone DNA known to have heavy withering syndrome infection (confirmed via histology and qPCR) to verify that these universal primer sets actually work.  I’m also using the withering syndrome primer sets on this DNA to function as a positive control.

Template DNA is: 09:20-08 (from tissue)

Background info for template DNA is here: Red/Pink/Pinto

Primers being used are:

  • 16s/23s-F/R
  • 27F, 1492R
  • EHR16D, EHR16R (universal ehrlichia)
  • EUB-A/B
  • 18s EUK 581 F, 18s EUK 1134 R
  • WSN1 (withering syndrome)

Master mix calcs are here: 20150204 – Ireland Clam Troubleshooting GoTaq Flexi

All samples were run in duplicate.

Cycling params were:
1 cycle of:

  • 95C – 10mins

40 cycles of:

  • 95C – 15s
  • 50C – 15s
  • 72C – 2mins

Ran samples out on a 0.8% agarose,  1x TBE gel w/EtBr

Results:

 

Nothing.  Since there’s nothing, I didn’t bother labelling the gel. So, this suggests that the PCR reactions aren’t working.  Will get newer reagents to replace the 5yr+ old reagents I have been using.  Also will try a different thermal cycler, just to rule out all possibilities.

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

Ran cPCR, but used Promega GoTaq Flexi instead of the 2x Immomix Taq (Bioline). Master mix calcs are here. cPCR recipe was provided by Lisa.

All samples run in duplicate.

Results:

Great, no amplification of any sort. More troubleshooting??!! Will begin troubleshooting tomorrow.

PCR – Repeat of RA Primers on LCM and Genomiphi DNA

Due to the failure of two consecutive attempts, I am repeating this PCR using the reagents and cycling params listed in Lisa’s notebook. Master mix calcs are here. Not noted in the master mix calcs are the MgCl2 and dNTP stock concentrations. They were 25mM and 10mM, respectively.

Cycling params:

  • 95C – 3mins

40 cycles of:

  • 95C – 1m
  • 62C – 30s
  • 72C – 30s
  • 72C – 10m

Positive Control: “1:100 Plasmid RLP” from 7/24/09. This was supplied by Lisa to use as a positive control for these primers.

Results:

Lane 1 – Hyperladder IV

Lane 2 – LCM Classic

Lane 3 – LCM New

Lane 4 – Genomiphi Classic

Lane 5 – Genomiphi New

Lane 6 – Positive Control

Lane 7 – NTC

It worked, finally! Both LCM samples clearly amplify. However, neither of the Genomiphi prepped samples produce any product. Will discuss with Lisa and/or Carolyn to see what they think about this and what they’ve seen before. But, it seems clear that the Genomiphi procedure did not work. Could be due to age of the kit (see 20110414 for notes on expiration date)