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 Convetional PCR Assay Validation (Repeatibility/Reproducibility)

This is an exact repeat of the run from 20121220. Ran conventional PCR using RA5-1 and RA3-6 primers as part of assay validation for the the abalone Withering Syndrome conventional PCR. Samples were low, medium and high concentrations of WS from fecal, tissue and water DNA extractions. The positive control used was the 30,000 copy from the WS standard curve plasmid made on 20120730. Master mix calcs are here.

Cycling params:

  1. 95C – 10mins

  2. 95C – 1min

  3. 62C – 30s

Repeat Steps 2 & 3, 41 times.

Run on the Eppendorf thermalcycler.

Due to small expected product size (~160bp), samples were run on a 1.2% agarose TBE gel.

Results:

PCR – Withering Syndrome Convetional PCR Assay Validation (Repeatibility/Reproducibility)

This is an exact repeat of the run from 20121220. Ran conventional PCR using RA5-1 and RA3-6 primers as part of assay validation for the the abalone Withering Syndrome conventional PCR. Samples were low, medium and high concentrations of WS from fecal, tissue and water DNA extractions. The positive control used was the 30,000 copy from the WS standard curve plasmid made on 20120730. Master mix calcs are here.

Cycling params:

  1. 95C – 10mins

  2. 95C – 1min

  3. 62C – 30s

Repeat Steps 2 & 3, 41 times.

Run on the Eppendorf thermal cycler.

Due to small expected product size (~160bp), samples were run on a 1.2% agarose TBE gel.

Results:

PCR – Withering Syndrome Convetional PCR Assay Validation (Repeatibility/Reproducibility)

This is an exact repeat of the run from 20121220. Ran conventional PCR using RA5-1 and RA3-6 primers as part of assay validation for the the abalone Withering Syndrome conventional PCR. Samples were low, medium and high concentrations of WS from fecal, tissue and water DNA extractions. The positive control used was the 30,000 copy from the WS standard curve plasmid made on 20120730. Master mix calcs are here.

Cycling params:

  1. 95C – 10mins

  2. 95C – 1min

  3. 62C – 30s

Repeat Steps 2 & 3, 41 times.

Run on the Eppendorf thermalcycler.

Due to small expected product size (~160bp), samples were run on a 1.2% agarose TBE gel.

Results:

PCR – Withering Syndrome Convetional PCR Assay Validation (Repeatibility/Reproducibility)

This is an exact repeat of the run from 20121220. Ran conventional PCR using RA5-1 and RA3-6 primers as part of assay validation for the the abalone Withering Syndrome conventional PCR. Samples were low, medium and high concentrations of WS from fecal, tissue and water DNA extractions. The positive control used was the 30,000 copy from the WS standard curve plasmid made on 20120730. Master mix calcs are here.

Cycling params:

  1. 95C – 10mins

  2. 95C – 1min

  3. 62C – 30s

Repeat Steps 2 & 3, 41 times.

Run on the MJ thermalcycler.

Due to small expected product size (~160bp), samples were run on a 1.2% agarose TBE gel.

Results:

PCR – Withering Syndrome Convetional PCR Assay Validation (Repeatibility/Reproducibility)

This is an exact repeat of the run from 20121217. Ran conventional PCR using RA5-1 and RA3-6 primers as part of assay validation for the the abalone Withering Syndrome conventional PCR. Samples were low, medium and high concentrations of WS from fecal, tissue and water DNA extractions. The positive control used was the 30,000 copy from the WS standard curve plasmid made on 20120730. Master mix calcs are here.

Cycling params:

  1. 95C – 10mins

  2. 95C – 1min

  3. 62C – 30s

Repeat Steps 2 & 3, 41 times.

Run on the MJ thermalcycler.

Due to small expected product size (~160bp), samples were run on a 1.2% agarose TBE gel.

Results:

PCR – Withering Syndrome Conventional PCR Assay Validation (Repeatibility/Reproducibility)

Ran conventional PCR using RA5-1 and RA3-6 primers as part of assay validation for the the abalone Withering Syndrome conventional PCR. Samples were low, medium and high concentrations of WS from fecal, tissue and water DNA extractions. The positive control used was 30,000 copy from the WS standard curve made on 20120730. Master mix calcs are here.

Cycling params:

  1. 95C – 10mins

  2. 95C – 1min

  3. 62C – 30s

Repeat Steps 2 & 3, 41 times.

Run on the MJ thermalcycler.

Due to small expected product size (~160bp), samples were run on a 1.2% agarose TBE gel.

Results:

Hyperladder V was used as molecular weight markers.

Plasmid Curve – RLP Plasmid Size Determination

After looking through Nate’s notebook and aligning the withering syndrome primers and probe to their source sequence (GenBank Accession AF133090; see below) I have discovered a few things:

  1. The vector that was used for cloning was Invitrogen’s PCR 4-TOPO (3956bp). This was found in Nate’s notebook #1 WS qPCR on page 3.

  2. The insert was generated with the primers RA 5-1 and RA 3-6. The resulting fragment size is 158bp. However, Nate’s notebook lists the size as 176bp.

  3. The size of the vector plus the insert should be 4114bp. However, since Nate used the incorrect insert size, he calculated the combine vector+insert size as 4133bp.

  4. Although this doesn’t seem to have much of an effect on the withering syndrome qPCR assay (based on the assay working for years prior to our current issues), the WSN1R primer sequence actually lies partially outside of the cloned region of AF133090. See the alignment below.

Made the dilutions for the RLP standard curve, based on a size of 4114bp. The calculations are here.

20120406 – UPDATE: Carolyn has just remembered that there are multiple, different versions of RLP cloned into a variety of vectors. After discussion with Carolyn and Robyn, Carolyn definitively determined that Lisa and I had not been using the correct clones with the correct RLP insert! The correct vector/insert should be p16RK3-C with the entire AF133090 sequence insert.

PCR – RA Primers on LCM DNA (from 20110413) and Clean Genomiphi DNA (from earlier today)

Ran PCR in exactly the same fashion as 20110428 to assess whether or not the clean up procedure improved our ability to perform a successful PCR on the Genomphi’d DNA. Master mix calcs are here.

Results:

From left to right:

Lane 1 – Hyperladder I (Bioline)

Lane 2 – LCM DNA Classic

Lane 3 – LCM DNA New

Lane 4 – Genomiphi Clean Classic

Lane 5 – Genomiphi Clean New

Lane 6 – Pos. Control

Lane 7 – NTC

Unfortunately, the clean up procedure didn’t resolve the lack of amplification issue that we’re seeing in the Genomiphi’d samples. Since we’ve started the process of enriching for rickettsia from abalone tissue, I’m not going to worry about further analysis of why the Genomiphi’d samples don’t work in PCR. Hopefully the enrichment procedure for rickettsia will yield significant amounts of bug to provide us with workable quantities of DNA.

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)