6 10 2015 Flanking Primer PCR pt. 2

Yesterday I ran a PCR using a couple flanking primers I created to see if they worked. The primers worked successfully, which you can read about here. Before the end of the day I ran the remaining 11 primers using the same protocol. Today I ran gels on all of the products to see which ones worked and isolate the PCR product for sequencing.

Primers

1672	Flk_PGRP_FWD	AGCTGGTGCAGTCCTATCAG	JH	6/1/2015	20		59	O.lurida	PGRP-S Flanking	O75594
1671	Flk_PGRP_REV	TGTGTATGAAAAGTAATGAAGAGCA	JH	6/1/2015	25		57	O.lurida		O75594
1670	Flk_CARM_FWD	TTCACACAGCCCATTGTGGAT	JH	6/1/2015	21		60	O.lurida	CARM1 Flanking	Q6DC04
1669	Flk_CARM_REV	TGGGATGGGTCAGATAAACCT	JH	6/1/2015	21		58	O.lurida		Q6DC04
1668	Flk_BMP2_FWD	GGCTGGCTGGATCGTCAT	JH	6/1/2015	18		60	O.lurida	BMP2 Flanking	P12643
1667	Flk_BMP2_REV	ATGGAGTCTGTGGACGGTTTG	JH	6/1/2015	21		60	O.lurida		P12643
1666	Flk_HSPb11_FWD	AGAATTGTCTGTGGAATCGAGC	JH	6/1/2015	22		59	O.lurida	HSPb11 Flanking	Q9Y547
1665	Flk_HSPb11_REV	ATCAACGCCAGGGGAACTTG	JH	6/1/2015	20		61	O.lurida		Q9Y547
1664	Flk_PGE/EP4_FWD	GCTCAACGAATTGCTCTACTCC	JH	6/1/2015	22		59	O.lurida	PGE/EP4 Flanking	P32240
1663	Flk_PGE/EP4_REV	TCCGTCTGCTTTTTAGAATGGTA	JH	6/1/2015	23		58	O.lurida		P32240
1662	Flk_GABABR_FWD	GAGGAGGACACGAAACTCCG	JH	6/1/2015	20		60	O.lurida	GABABR Flanking	Q9WV18
1661	Flk_GABABR_REV	TGCACCACACTCCTGATGAC	JH	6/1/2015	20		60	O.lurida		Q9WV18
1660	Flk_GRB2_FWD	TCAGAACTGGTTCAAAGCTGAGT	JH	6/1/2015	23		60	O.lurida	GRB2 Flanking	P62994
1659	Flk_GRB2_REV	ACTGCGCTGACATACTGGAC	JH	6/1/2015	20		60	O.lurida		P62994
1658	Flk_H3.3_FWD	CCAATGACAAATGAGCCACACAA	JH	6/1/2015	23		60	O.lurida	H3.3 Flanking	Q6P823
1657	Flk_H3.3_REV	TCGTACAAAGCAAACTGCACG	JH	6/1/2015	21		60	O.lurida		Q6P823
1656	Flk_H2A.V_FWD	GCGATGGAGTTGATGAGGTG	JH	6/1/2015	20		59	O.lurida	H2A.V Flanking	P08991
1655	Flk_H2A.V_REV	CAAGGCAGTTTCTCGTTCGG	JH	6/1/2015	20		59	O.lurida		P08991
1654	Flk_H2A_FWD	TGCTGGGGTTTTTCTGGGTC	JH	6/1/2015	20		60	O.lurida	H2A Flanking	P02270
1653	Flk_H2A_REV	TCAGGACGTGGTAAAGGAGGA	JH	6/1/2015	21		60	O.lurida		P02270
1652	Flk_p29ING_FWD	GTGGACACACATGCACTCCT	JH	6/1/2015	20		60	O.lurida	p29ING4 Flanking	Q8C0D7
1651	Flk_p29ING_REV	AAGCAGACTCAGATTCAGGC	JH	6/1/2015	20		58	O.lurida		Q8C0D7

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 (ul)	Final Concentration
2x Apex Red	12.5	125
Forward Primer (10uM)	0.5	5
Reverse Primer (10uM)	0.5	5
H20	10.5	105
1:2 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	175-200 ml
Agarose	2.3-2.6 g
EtBr	17.5-20 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 gels at 100 v for 35 minutes. I placed it on the transilluminator to view any bands that may have formed. The gels are below. 

Gel 1 layout:

PGRP-S

CARM

BMP2

Ladder

NT1

HT1

ST1

NC1

HC1

SC1

NTC

Ladder

NT1

HT1

ST1

NC1

HC1

SC1

NTC

Ladder

NT1

HT1

ST1

BMP2

HSPb11

PGE/EP4

NC1

HC1

SC1

NTC

Ladder

NT1

HT1

ST1

NC1

HC1

SC1

NTC

Ladder

NT1

HT1

ST1

NC1

HC1

SC1

NTC

Gel 2 layout:

GABABR

GRB2

H3.3

Ladder

NT1

HT1

ST1

NC1

HC1

SC1

NTC

Ladder

NT1

HT1

ST1

NC1

HC1

SC1

NTC

Ladder

NT1

HT1

ST1

H3.3

H2A.V

H2A

NC1

HC1

SC1

NTC

Ladder

NT1

HT1

ST1

NC1

HC1

SC1

NTC

Ladder

NT1

HT1

ST1

NC1

HC1

SC1

NTC

p291N4

Ladder

NT1

HT1

ST1

NC1

HC1

SC1

NTC

Ladder

Empty

Empty

Empty

Empty

Empty

Empty

Empty

Empty

Empty

Empty

Empty

In Gel 1, the PGRP-S flanking primer failed completely. Also the Oyster Bay heat treated samples failed to amplify with the CARM and PGE/EP4 flanking primers. These did amplify previously so I'm not sure what it means. 

In Gel 2, The Heat treated Dabob sample failed to amplify with the GABABR primer again not sure what it means. Interesting, the Fidalgo heat treated sample's PCR product in the H3.3 region has a much higher molecular weight. Steven suggested this could be an alternative splicing which would be interesting. 

All bands except those in the failed PGRP primer were cut out and placed in either labelled 1.5 ml tubes or labelled Millipore Purification columns. The ones in the column were centrifuged for 10 minutes at 5000 rcf. The purified solutions and gel bits are stored in the 4 C fridge in 209. When we get more purification columns either I or Sam will spin down the remaining bands. Once the samples are isolated they will be sent off for sequencing. Hopefully the sequences will be the same in all populations so that the qPCR's can be trusted. That or they are so wildly different they expose significant differences in the genes between populations. Either way it should be interesting. 

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. 

5 29 2015 Designing Primers for Sequencing Target areas

Today after some conversations between Brent and Steven based on the previous primer checks (here and here) the idea arose that not all populations may share the same isoforms of the target genes. To determine if the genes are the same in all populations, its necessary to produce a sequence containing the primer region plus 100 bp on either side for sequencing in sub samples from each population. This put me in a unique situation. I needed to not only have the primer for the target gene, but also a primer that encompasses the target gene + original primer + 100 bp on either side of the sequence. I decided to use the NCBI Primer Blast, that I used to generate the original primers, to create the new primers.

First I need to figure out where in the sequence does the original primer amplify. To do this I have to upload the original sequence and the primer sequences into NCBI. For this I'm looking at the TLR2.1 primer since it has the most interesting results from the primer check.

The original primer sequences for TLR2.1:
FWD ACAAAGATTCCACCCGGCAA

REV  ACACCAACGACAGGAAGTGG

Product Size  109 bp

The original sequence for target gene:

tcaaaacagattttggagtgtaacgtctttaaaaattactaatacagcaacaatgaaatcattttgccatgaattgcttcacgatttgccagaacagcttctggccatttctattcattgtccactctacatgagatgctgacttgagaagcaccttcagagattgattaatatgttcagaacgaatatcttgtaatatgatcagcaatagcttgtcaccgcctccgtcagccaaagtgtggttggcaatggcggcttcgtacttacaccattgatcgtccaagaaattattggacagcaccaaaatgaattttctgctgacttcaatgctttccagaaagtcatccacaaagattccacccggcaagatatctctctcgtgcaaacaaagcttgtacttgtttctctctaccatctgaacaagttcagacattatccacttcctgtcgttggtgttgtaagcaacaaaaccatcatagaggaattcatcatctgtaaatcttttataccccatgggttttctgtttttgcatgtgtatatataatacttaatgtcccaacgaaactttctcaacagaacaatggtcaaaatcaccaaggacactaatgcaaacgaagcgcatactagaatgacataggggcttattttgtgacattcggacgcaggatcaaagtgtgttatactttttcctttcaagtctgacggggaagcacagatgtattgatgaggatatccaacagtcctattttggttgttttcaacccagagtcggaaccattccagttggcatccacagtccaatggattcaaggacacatcaattcgaaaatcttttttcttccaaaggaaagcaggcagagaagattcattgatactgctcaaacgattgccacgaaaaatcaattcattcaatctagtcaaattttgaacaccgctctttcgaatttctgttattcggttaacactgtagtcaacggacgttagctgtgaaaaatcagtcagtgtagtcatttgatcgatttcgccattaacaatagccaaaactgacagatcctttagaggtgatagcatctctgcaacatcagtgttggatagatcggtgttcattattttcaatgcttttatatttctacattcagaaaagatgtatttgaagcctcctacgcttttttccaccatacgtccgatagagagtgttcggagggaattggatgataatgacctaccatctaaagcgactccatgcaagttatcaatgatcaaagtttgcaaacgattcatattcaaaaaggaatacaaggaaatgtcatgaattgatgtgtgtgaaacattaaaaaattcaagggatttcaggcagtagccctcttgtatgaaatttgttgtttcccgaataccattcactgccaggtgcaattgttgcaagtttgggaaggcggctatacgtttctcattggataaacaaaactcgggaatcttctcaaaagaattgttagtaaaatacagttcttttaaagaaggaactgagctatttggcaatttataccttgagattaggttgcttcctaaatcgattttgtggatattgattaatttagcgaaggatttaaaag

First I run the sequence and the primers through NCBI Primer Blast:

Once the Primer Blast returns the primer target. I can then find where the primer begins and ends in the original sequence.

The forward primer begins at roughly 340 bp and the reverse primer begins at roughly 460 bp. Using these numbers I subtract 100 bp from the start of the forward primer and add 100 bp to the beginning of the reverse primer. This extends the range to 240 bp - 560 bp which encompasses the original target and primer area as well as 100 bp on either side. To give the Primer Blast some leeway to produce the new primers, I give a 100 bp range for the forward and reverse primers. The range for the forward primer becomes 140-240 bp. The range for the reverse primer becomes 560-660 bp. I also required the product size to be between 325 bp and 1000 bp long. This is so that the 100 bp on either side plus the 109 bp original target are amplified.

The primers that NCBI now covers the entire area that needs to be sequenced. All I have to do is choose primers that have low self complimentarity.

After looking at the offered primers I chose one that should give a 425 bp product with low self complimentarity.

This is the quickest way I've found to take the primers I produced originally and create primers to sequence the target area of interest. If this works I'll do this with all the primers of interest that worked in the previous primer runs.