Tag Archives: luciferase

PCR – Sea Pen luciferase

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:

 

 

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.

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