Jake Heare Research Central

Wednesday, June 10, 2015

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

Add agarose to TAE.
Microwave 1 minute stir
Repeat until no particulate matter in solution
Add EtBr while agarose still hot
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

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.

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:

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

Add agarose to TAE.
Microwave 1 minute stir
Repeat until no particulate matter in solution
Add EtBr while agarose still hot
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.

6 9 2015 Reverse Engineering a Flanking Primer

Today I needed to develop a way to reverse engineer a flanking primer from a primer that someone else designed. For my qPCR runs, I have an HSP70 and Actin primers which were developed previously by someone else with little information on how they were developed. The primers work great but to determine if they are reaching the same efficiency in every sample or cover the same isoform of the gene in every population I need to develop flanking primers. Luckily NCBI has some advance functions in the Primer Blast program which allowed me to find the template region in my transcriptome to develop flanking primers.

First I needed to take the sequences of the primers I had and find where they existed in the Transcriptome I'm using.

Under primer parameters I put in the forward and reverse primer sequences. Then under Primer Pair Specificity Checking Parameters I changed the Database type to custom and uploaded the Oly Transcriptome V3. Then for added effect I changed the organism to the Ostrea taxa. I don't think this really did much but it didn't detract either. Then I hit get primers and waited for the results.

You can see what the input looks like above. Below is the output for the primers.

This out put tells me the reference name of the template from the Transcriptome on the left, the size of the primer product, and where the primer lays down on the sequence. Under each primer sequence there is a line of ...................... these indicate that the complimentary strand is a 100% match. If any of these dots are replaced with a letter it tells you whats different and that its not a 100% match. You can also see that the top 3 template matches have reference names that are very similar. This is because they are overlapping reads of the same region. If the names were different then we could assume it was a different region.

Once you've got the reference name, then you can follow the same method I used to develop the other flanking primers here.

Monday, June 1, 2015

June 2015 Goals

So before I state what this month's goals are, I will briefly mention how I did on last months goals.

May 2015 Goals

Process samples to produce cDNA for qPCR
Run qPCR using primers for HSP70, Actin, and other interesting primers
Edit the Materials and Methods section
Write the Results sections of my next manuscript
Begin writing the Intro/Discussion
Prepare for my wedding

How I did May 2015:

1. I processed the 310 samples but Sam White processed my samples. There hasn't been cDNA made from all of them but that's okay cause I'm still testing primers.

2. I ran test qPCR's for HSP70, Actin, and the other primers I generated. Some worked, some didn't. I haven't been able to do the formal qPCR run due to some complications of population differences.

3. I have made minor edits to the Materials and Methods but no where near what they should be. Will be work on that soon.

4. I have no results as of yet.

5. I cannot begin with Intro/Discussion

6. I have prepared a ton for the wedding.

June 2015 Goals

Run qPCR tests with chosen flanking primers and sequence expression

Run qPCR tests with chosen primers to reflect populations level differences

Edit Materials/Methods

Write Results

Have committee meeting

Write Intro/Discussion

Get Married!