6 25 2015 Flanking PCR pt 4

Yesterday I ran a PCR for some flanking primers that failed to get good coverage based on Steven's analysis. Today I ran the products on a gel and excised the bands for sanger sequencing.

Primers:

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
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

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
Template	1

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 25 ul of each product on a 1.3% agarose gel

Gel Reagent Table

Reagent	Volume
1X Low TAE	175 ml
Agarose	2.3 g
EtBr	17.5 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 40 minutes. I placed it on the transilluminator to view any bands that may have formed. In my haste I forgot to image the gel, Luckily the gel ran fine with strong banding and minor streaking due to over filled wells. 

After running the gel I excised the bands. Once the bands were cut out of the gel and placed in a Millipore Purification Column and centrifuged at 5,000 rcf for 10 minutes to collect the purified DNA. 

To send them off for sequencing I plated 4 replicates of 10 ul for each sample in the appropriate tube. On a second plate I plated two replicates of 3 uM primer working stock for each appropriate sample. These plates are now being held in the 4 C in 213 for sequencing. 

6 23 2015 Flanking Primer PCR pt. 3

Today I ran a PCR on the two new flanking primers Actin and HSP70 as well as re run some primers that failed to sequence (HSPb11, PGEEP4, BMP2). After running PCR, I ran the products out on gels and then extracted the bands for sequencing.

Primers:

1680	Flk_HSP70_FWD	GACCCGGATGTCCAGAGGAA	JH	6/9/2015	20		60	O.lurida	HSP70 Flanking
1679	Flk_HSP70_REV	TCAGGACGGCTTGTGAACG	JH	6/9/2015	19		60	O.lurida
1678	Flk_Actin_FWD	TCGAGGGAGGAAGAGGAAGC	JH	6/9/2015	20		59	O.lurida	Actin Flanking
1677	Flk_Actin_REV	AACTGGGACGACATGGAGAA	JH	6/9/2015	20		61	O.lurida
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
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

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
Template	1

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 25 ul of each product on a 1.3% agarose gel

Gel Reagent Table

Reagent	Volume
1X Low TAE	175 ml
Agarose	2.3 g
EtBr	17.5 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 40 minutes. I placed it on the transilluminator to view any bands that may have formed. The gel is below

Gel Layout:

Actin

HSP70

Ladder

HC1

NC1

SC1

HT1

NT1

ST1

NTC

Ladder

HC1

NC1

SC1

HT1

NT1

ST1

NTC

Empty

Empty

Empty

Empty

PGEEP4

HSPb11

Ladder

HC1

NC1

SC1

HT1

NT1

ST1

NTC

Ladder

HC1

NC1

SC1

HT1

NT1

ST1

NTC

Empty

Empty

Empty

Empty

BMP2

Ladder

HC1

NC1

SC1

HT1

NT1

ST1

NTC

Empty

Empty

Empty

Empty

Empty

Empty

Empty

Empty

Empty

Empty

Empty

Empty

It appears the Actin flanking primer failed hard. The HSP70 primer seemed to do pretty well but still created several smaller bands. I carefully extracted only the largest band from the HSP70 samples to ensure that it was the right product. The rest of the primers worked like the had previously. 

Once the bands were cut out of the gel and placed in a Millipore Purification Column and centrifuged at 5,000 rcf for 10 minutes to collect the purified DNA. 

To send them off for sequencing I plated 4 replicates of 10 ul for each sample in the appropriate tube. On a second plate I plated two replicates of 3 uM primer working stock for each appropriate sample. These plates are now being held in the 4 C in 213 for sequencing. 

Sam's last sequencing came back today and it appears there are more primers that need to be re amplified and sequenced I will work on that tomorrow. I will also make sure to send off the TLR2.1 samples as they were overlooked in the last batch.  

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.