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:

1676	Flk_TLR_FWD	GCAATAGCTTGTCACCGCC	JH	6/1/2015	20		59	O.lurida	TLR2.1 Flanking	Q9DD78
1675	Flk_TLR_REV	TCTAGTATGCGCTTCGTTTGC	JH	6/1/2015	20		59	O.lurida		Q9DD78
1674	Flk_CRAF_FWD	GGACATCCAGTGGCAACATTC	JH	6/1/2015	21		60	O.lurida	CRAF1 Flanking	Q60803
1673	Flk_CRAF_REV	CCAGGACATTAGGCTTGCTGA	JH	6/1/2015	21		60	O.lurida		Q60803

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

Reagent Table

Reaction_Components	Volume	Final Concentration
2x Apex Red	12.5	125
Forward Primer (10uM)	0.5	5
Reverse Primer (10uM)	0.5	5
H20	10.5	105
1:1 cDNA	1
	25

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:

Temp	Time
95 C	5 min
95 C	30 sec
55 C	30 sec
72 C	30 sec
repeat steps 2-4 40 times
72 C	3 min
4 C	Hold

Once the PCR finished I ran the products on a 1.3% agarose gel

Gel Reagent Table

Reagent	Volume
1X Low TAE	100 ml
Agarose	1.3 g
EtBr	10 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 1

Empty

Ladder

NTC

NT1

HT1

ST1

NC1

HC1

SC1

NTC

Ladder

NTC

NT1

HT1

ST1

NC1

HC1

SC1

NTC

Ladder

Empty

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.