---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
date: 2015-07-02 22:31:44+00:00
layout: post
slug: pcr-sea-pen-luciferase
title: PCR - Sea Pen luciferase
categories:
  - 2015
  - Miscellaneous
tags:
  - Apex Red Master Mix
  - gDNA
  - gel
  - luciferase
  - O'GeneRuler 100bp DNA ladder
  - PCR
  - Renilla reniformis
  - sea pen
---

Ran a PCR to obtain luciferase DNA for sequencing.

Used sea pen [gDNA extracted by Jonathan on 20150527](https://jda26.blogspot.com/2015/05/05272015-started-dna-extraction-on.html).

Primers:

<table cellpadding="0" cellspacing="0" border="1" dir="ltr" > 
<tbody >
<tr >

<td data-sheets-value="[null,3,null,1604]" >SRID
</td>

<td data-sheets-value="[null,2,"Rr_46_65F"]" >Name
</td>
</tr>
<tr >

<td data-sheets-value="[null,3,null,1604]" >1604
</td>

<td data-sheets-value="[null,2,"Rr_46_65F"]" >Rr_46_65F
</td>
</tr>
<tr >

<td data-sheets-value="[null,3,null,1603]" >1603
</td>

<td data-sheets-value="[null,2,"Rr_887_868R"]" >Rr_887_868R
</td>
</tr>
</tbody>
</table>

Master mix calcs are here: [20150702_seapen_PCR](20150702_seapen_PCR_calcs)

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)(https://github.com/sr320/LabDocs/blob/master/protocols/Commercial_Protocols/ThermoFisher_OGeneRuler100bpDNA_ladder.jpg?raw=true)[![](http://eagle.fish.washington.edu/Arabidopsis/20150702_seapen_PCR_gel.jpeg)](http://eagle.fish.washington.edu/Arabidopsis/20150702_seapen_PCR_gel.jpeg)

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.
