Ran a PCR to obtain luciferase DNA for sequencing.

Used sea pen gDNA extracted by Jonathan on 20150527.

Primers:

Master mix calcs are here: 20150702_seapen_PCR

Cycling params:

1. 95C – 10mins
2. 95C – 15s
3. 55C – 15s
4. 72C – 1min
5. Go to Step 2 39 times

Ran samples on 0.8% agarose, low TAE gel stained with EtBr.

Results:

Loading:

Lane 1 – ladder

Lane 2 – empty

Lane 3 – sea pen gDNA

Lane 4 – NTC

PCR did not work. Was expecting a band of ~800bp.

Looks like I may have overloaded the PCR reaction with gDNA. Used 10μL of gDNA.

However, that is quite the smear, suggesting a significant amount of degradation present in the gDNA.

Will re-run this PCR next week with less gDNA (or, cDNA instead) in order to generate a PCR product.

Lexie’s PCR with this primer set and a pool of Mercenria cDNA has yielded contamination in all of her waters. Performed two sets of PCR: one with her existing primer working stocks and the other with a fresh aliquot of primer working stocks. Used my own reagents/water. PCR set up and cycling params are here. PCR ran O/N.

Results:

Lane 1 – 100bp ladder

Lane 2 – H2O (Lexie’s primer stocks)

Lane 3 – H2O (Lexie’s primer stocks)

Lane 4 – cDNA (Lexie’s primer stocks)

Lane 5 – H2O (fresh primer stocks)

Lane 6 – H2O (fresh primer stocks)

Lane 7 – cDNA (fresh primer stocks)

All water samples look clean and there’s a nice bright band in the cDNA samples. Lexie’s contamination issue is probably a technique issue and not one of reagent contamination.