Tuesday, June 9, 2015

6 9 2015 Flanking Primer Trial PCR

Today I ran a trial PCR on subsample of the flanking primers to see if they worked correctly. The primers I used were:

1676Flk_TLR_FWDGCAATAGCTTGTCACCGCCJH6/1/20152059O.luridaTLR2.1 FlankingQ9DD78
1674Flk_CRAF_FWDGGACATCCAGTGGCAACATTCJH6/1/20152160O.luridaCRAF1 FlankingQ60803

Using the Apex Red PCR Master Mix I created master mixes for each set of primers and ran them together. 

Reagent Table

Reaction_ComponentsVolumeFinal Concentration
2x Apex Red12.5125
Forward Primer (10uM)0.55
Reverse Primer (10uM)0.55
1:1 cDNA1
Using the final concentration I mixed each master mix going from largest to smallest volume. 
I then pipetted 24 ul in each well followed by 1 ul of water or sample depending. 

Then I ran the following PCR program:

95 C5 min
95 C30 sec
55 C30 sec
72 C30 sec
repeat steps 2-4 40 times
72 C3 min
4 CHold
Once the PCR finished I ran the products on a 1.3% agarose gel

Gel Reagent Table
1X Low TAE100 ml
Agarose1.3 g
EtBr10 ul 

  1. Add agarose to TAE.
  2. Microwave 1 minute stir
  3. Repeat until no particulate matter in solution
  4. Add EtBr while agarose still hot
  5. Gently pour in one corner of the gel cast until tray is full 
I then ran the gel at 100 v for 35 minutes. I placed it on the transilluminator to view any bands that may have formed. The gel is below. 

Gel Layout:
TLR2.1 CRAF 1Empty

As you can tell the TLR2.1 primers still did not amplify in the Dabob population. This could be an indication that this gene is not expressed in this population. More population replicates are needed to determine if this is true. 

After seeing these nice bands, I cut them out and stored the individual bands for sequencing in 1.5 ml tubes. I also decided to run the remaining primers using the reagents above and then produce the gels tomorrow. 


  1. Bands! Excellent!

    One thing I noticed is that your gel layout guide only has 17 entries, but your gel has 20 wells, so the gel layout guide doesn't match up correctly with the gel.

  2. Nice catch. Somehow I forgot to put in the SC group on the layout.