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

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.