PCR - Sea Pen luciferase

2015
Apex Red Master Mix
gDNA
gel
luciferase
O’GeneRuler 100bp DNA ladder
PCR
Renilla reniformis
sea pen
Author

Sam White

Published

July 2, 2015

Ran a PCR to obtain luciferase DNA for sequencing.

Used sea pen gDNA extracted by Jonathan on 20150527.

Primers:

SRID Name
1604 Rr_46_65F
1603 Rr_887_868R

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:

(https://github.com/sr320/LabDocs/blob/master/protocols/Commercial_Protocols/ThermoFisher_OGeneRuler100bpDNA_ladder.jpg?raw=true)

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.