qPCR – LCM DNA

Ran three primer sets on laser capture microscopy (LCM) DNA samples from 2005 and 2007. Ran the following primer sets:

  • WSN1 (detects RLO)
  • RLOv_helicase (detects RLO phage)
  • XenoCal_prophage

The DNA samples were provided to me by Lisa. I’m not entirely sure of their history:

 

Master mix calcs (Google Sheets):

All samples were run in duplicate. Plate layout, cycling params, etc. are in the qPCR Reports (see Results below).

Standard curves:

Baseline threshold was manually set to 580 for the WSN1 samples, as previously determined by Lisa for this assay.

Baseline threshold was manually set to 580.5 for the RLOv DNA helicase samples, as previously determined by me on 20160128.

 

Results:

WSN1:

 

RLOv DNA helicase:

 

XenoCal prophage:

 

Summary table of all three genes in each sample. Unfortunately, I don’t fully understand the sample name nomenclature, so I can’t really come to any conclusions about the data. Will pass along to Carolyn, Lisa, and Stan.

It’s also important to note that, due to low sample volume, I did not quantify these samples. This is important because any samples listed below that are negative for all three genes can not be conclusively declared “negative”, since we can’t rule out the possibility that they simply lack any DNA.

Presumably they were quantified after their initial extraction?

SAMPLE WSN1 RLOv DNA HELICASE XC PROPHAGE
LCM New RLO 09 + + +
LCM ST RLO 09 - - -
LCM New 08:30-5 B + + +
LCM New 08:30-5 - - -
LCM ST 08:30-3 - - -
LCM WS RLO + - +

 

STANDARD, AMPLIFICATION, & MELT CURVES

 

WSN1

 

 


 

 

RLOv_DNA_helicase

 


 

 

XenoCal prophage

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.

Genomiphi Cleanup – Clean Up of Genomiphi’d LCM DNA (from 20110414)

According to a paper Lisa came across recently (REFERENCE), those researchers were unable to successfully perform PCR on samples that they had used the Genomphi V2 Kit (GE LifeSciences) on. As such, they mentioned that they had to use a PCR clean up kit after Genomiphi treatment in order to get subsequenct PCRs to work.

Used the QIAquick PCR Cleanup Kit (Qiagen) according to the manufacturer’s protocol.

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)

PCR – RA Primers on LCM and Genomiphi DNA (from 20110413 and 20110414)

This is a repeat of the PCR from 20110421, due to no amplification. This time I will be using the “RLP4″ protocol on the MJ thermalcycler. Master mix calcs are here.

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:

Absolutely no amplification of any sample (including positive control), even with the different cycling parameters. Will talk to Lisa and try using the same reagents as she’s used in the past (i.e. not use Immomix…).

I’m also curious as to why the ladder looks so crappy. My guess is that the gel is drifting a bit while running because it’s being run in the large gel box…

PCR – RA Primers on LCM and Genomiphi DNA (from 20110413 and 20110414)

Ran conventional PCR using RA 5-1 and RA 3-6 primers on the LCM and Genomiphi’d DNA isolated 20110413 and 20110414, respectively. Master mix calcs and cycling params are here.

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:

Well, the gel is totally and completely blank. Not even the positive control showed up. Will try to figure out what went wrong…

–UPDATE– Found original cycling parameters for this PCR in one of Lisa’s notebooks. Will try those when I repeat this.

DNA Amplification – LCM DNA from yesterday

Used the Genomiphi V2 Kit (Note: Expiration date on box listed as 05 Feb 2010!) according to the manufacturer’s protocol on both LCM DNAs extracted yesterday (New 08-1-702.17.10; Class 08:13-5a 2.17.10). Briefly, combined 1uL of LCM DNA with 9uL of Sample Buffer. Incubated 95C 3mins and then placed on ice. Added 9uL of Reaction Buffer and 1uL of Enzyme Mix to each sample. Incubated @ 30C for 1.5hrs, heated @ 65C for 10mins and then stored Genomiphi’d LCM DNA @ -20C.

DNA Extraction – LCM Samples from 20100217

Used the Pico Pure DNA Extraction Kit to extract DNA from laser capture microdissection samples (provided by Lisa; stored in -20C in Rm. 236) from 20100217 (New 08-1-702.17.10; Class 08:13-5a 2.17.10) according to the manufacturer’s protocol. Briefly, a single aliquot of lyophilized Proteinase K was reconstituted in 155uL of the provided buffer. 50uL of this solution was added to two fresh 0.5mL snap cap tubes. The LCM caps containing were placed on these 0.5mL tubes, inverted and tapped to cover them in the Proteinase K solution. Tubes were incubated ~24hrs @ 65C.