Showing posts with label Stress Experiment. Show all posts
Showing posts with label Stress Experiment. Show all posts

Tuesday, June 23, 2015

6 22 2015 Actin qPCR

Last night I ran a qPCR on Actin using the 48 cDNA samples produced by Sam. I ran it using the same procedure as I did for the CARM samples yesterday morning so that I can use it as a normalizing gene.

Primer Used:
1505Ol_Act_FGACCAGCCAAATCCAGACGABC6/13/2012205560O.luridaActin, adductor muscle
1504Ol_Act_RCGGTCGTACCACTGGTATCG6/13/2012206060O.luridaActin, adductor muscle 

Master Mix Reagent Table:
VolumeReactions X58
Ssofast Evagreen MM 10580
FWD Primer0.529
REV Primer0.529
Nuclease Free H2O8464
cDNA1
  1. Added reagents from greatest to least volume
  2. Vortexed
  3. Centrifuged briefly
  4. Pipetted 19 ul Master Mix to each tube
  5. Pipetted appropriate cDNA sample to each tube
  6. Centrifuged plate at 2000 rpm for 1 minute
  7. Ran Program Below
qPCR Program:
Sybr New Plate+Sybr cDNA 60 melt 2 Read
StepTemperatureTime
Initiation95 C10 min
Elongation95 C30 sec
60 C1 min
Read
72 C30 sec
Read
Repeat Elongation 39 times
Termination95 C1 min
55 C1 sec
Melt Curve Manual ramp 0.2C per sec Read 0.5 C55 - 95 C30 sec
21 C10 min
End
Plate Layout:
1234567
DNased 42215 HC1DNased 42215 NC1DNased 42215 SC1DNased 42215 HT1 1DNased 42215 NT1 1DNased 42215 ST1 1NTC
DNased 42215 HC2DNased 42215 NC2DNased 42215 SC2DNased 42215 HT1 2DNased 42215 NT1 2DNased 42215 ST1 2NTC
DNased 42215 HC3DNased 42215 NC3DNased 42215 SC3DNased 42215 HT1 3DNased 42215 NT1 3DNased 42215 ST1 3NTC
DNased 42215 HC4DNased 42215 NC4DNased 42215 SC4DNased 42215 HT1 4DNased 42215 NT1 4DNased 42215 ST1 4NTC
DNased 42215 HC5DNased 42215 NC5DNased 42215 SC5DNased 42215 HT1 5DNased 42215 NT1 5DNased 42215 ST1 5
DNased 42215 HC6DNased 42215 NC6DNased 42215 SC6DNased 42215 HT1 6DNased 42215 NT1 6DNased 42215 ST1 6
DNased 42215 HC7DNased 42215 NC7DNased 42215 SC7DNased 42215 HT1 7DNased 42215 NT1 7DNased 42215 ST1 7
DNased 42215 HC8DNased 42215 NC8DNased 42215 SC8DNased 42215 HT1 8DNased 42215 NT1 8DNased 42215 ST1 8
Results:

Amplification

 Melt Curve

As you can see the amplification and melt curves look good. Upon closer examination I did notice however that the rows at the top and bottom had much lower amplification than those in the middle. This is concerning as it could be giving me false values for samples in these spots. I've decided to forego using the Opticon and have now run samples on the BioRad in the Friedman lab in FSH. 

You can see the raw data file here.
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Monday, June 22, 2015

6 22 2015 CARM1 qPCR

Today I ran a qPCR for CARM1 using cDNA created by Sam last week. On friday Steven got the initial run of sequenced samples to check that all populations produced the same sequence. He selected CARM1 as being the most similar with the other primers having one issue or another. I put together a qPCR plate with all the cDNA samples to see how CARM1 differs between populations and treatment/control.

Primer Used:
1642CARM1_FWDTGGTTATCAACAGCCCCGACJH5/21/20152055O.luridaHistone-arginine methyltransferase CARM1 (EC 2.1.1.-) (EC 2.1.1.125) (Coactivator-associated arginine methyltransferase 1) (Protein arginine N-methyltransferase 4)Q6DC04
1641CARM1_REVGTTGTTGACCCCAGGAGGAGJH5/21/20152055O.luridaHistone-arginine methyltransferase CARM1 (EC 2.1.1.-) (EC 2.1.1.125) (Coactivator-associated arginine methyltransferase 1) (Protein arginine N-methyltransferase 4)Q6DC04

qPCR Master Mix Reagent Table:
VolumeReactions X55
Ssofast Evagreen MM 10550
FWD Primer0.527.5
REV Primer0.527.5
Nuclease Free H2O8440
cDNA1
  1. Added reagents from greatest to least volume
  2. Vortexed
  3. Centrifuged briefly
  4. Pipetted 19 ul Master Mix to each tube
  5. Pipetted appropriate cDNA sample to each tube
    1. Due to pipette error only 3 NTCs were run
  6. Centrifuged plate at 2000 rpm for 1 minute
  7. Ran Program Below
Sybr New Plate+Sybr cDNA 60 melt 2 Read
StepTemperatureTime
Initiation95 C10 min
Elongation95 C30 sec
60 C1 min
Read
72 C30 sec
Read
Repeat Elongation 39 times
Termination95 C1 min
55 C1 sec
Melt Curve Manual ramp 0.2C per sec Read 0.5 C55 - 95 C30 sec
21 C10 min
End
Plate Layout:
1234567
DNased 42215 HC1DNased 42215 NC1DNased 42215 SC1DNased 42215 HT1 1DNased 42215 NT1 1DNased 42215 ST1 1NTC
DNased 42215 HC2DNased 42215 NC2DNased 42215 SC2DNased 42215 HT1 2DNased 42215 NT1 2DNased 42215 ST1 2NTC
DNased 42215 HC3DNased 42215 NC3DNased 42215 SC3DNased 42215 HT1 3DNased 42215 NT1 3DNased 42215 ST1 3NTC
DNased 42215 HC4DNased 42215 NC4DNased 42215 SC4DNased 42215 HT1 4DNased 42215 NT1 4DNased 42215 ST1 4
DNased 42215 HC5DNased 42215 NC5DNased 42215 SC5DNased 42215 HT1 5DNased 42215 NT1 5DNased 42215 ST1 5
DNased 42215 HC6DNased 42215 NC6DNased 42215 SC6DNased 42215 HT1 6DNased 42215 NT1 6DNased 42215 ST1 6
DNased 42215 HC7DNased 42215 NC7DNased 42215 SC7DNased 42215 HT1 7DNased 42215 NT1 7DNased 42215 ST1 7
DNased 42215 HC8DNased 42215 NC8DNased 42215 SC8DNased 42215 HT1 8DNased 42215 NT1 8DNased 42215 ST1 8

Results:

HC1-8 Amplification


 HC1-8 Melt Curve
NC1-8 Amplification
 NC1-8 Melt Curve
SC1-8 Amplification
 SC1-8 Melt Curve
HT1-8 Amplification
 HT1-8 Melt Curve
NT1-8 Amplification
 NT1-8 Melt Curve
ST1-8 Amplification
 ST1-8 Melt Curve
NTC 1-3 Amplification
 NTC 1-3 Melt Curve

As you can see there is good amplification amoung all samples but without running the data through PCR Miner I can't distinguish a difference. Also there was some amplification in the NTC but it appears to be nonspecific with a much higher melting temp than the actual products. These high temp amplifiers don't appear in any of the other samples so I'm not worried about them as possible contamination. I've run a PCR on some more flanking primers to have them sequenced and am running a qPCR for Actin to see if we have similar results.

You can see the raw data file here.
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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

1672Flk_PGRP_FWDAGCTGGTGCAGTCCTATCAGJH6/1/20152059O.luridaPGRP-S FlankingO75594
1671Flk_PGRP_REVTGTGTATGAAAAGTAATGAAGAGCAJH6/1/20152557O.luridaO75594
1670Flk_CARM_FWDTTCACACAGCCCATTGTGGATJH6/1/20152160O.luridaCARM1 FlankingQ6DC04
1669Flk_CARM_REVTGGGATGGGTCAGATAAACCTJH6/1/20152158O.luridaQ6DC04
1668Flk_BMP2_FWDGGCTGGCTGGATCGTCATJH6/1/20151860O.luridaBMP2 FlankingP12643
1667Flk_BMP2_REVATGGAGTCTGTGGACGGTTTGJH6/1/20152160O.luridaP12643
1666Flk_HSPb11_FWDAGAATTGTCTGTGGAATCGAGCJH6/1/20152259O.luridaHSPb11 FlankingQ9Y547
1665Flk_HSPb11_REVATCAACGCCAGGGGAACTTGJH6/1/20152061O.luridaQ9Y547
1664Flk_PGE/EP4_FWDGCTCAACGAATTGCTCTACTCCJH6/1/20152259O.luridaPGE/EP4 FlankingP32240
1663Flk_PGE/EP4_REVTCCGTCTGCTTTTTAGAATGGTAJH6/1/20152358O.luridaP32240
1662Flk_GABABR_FWDGAGGAGGACACGAAACTCCGJH6/1/20152060O.luridaGABABR FlankingQ9WV18
1661Flk_GABABR_REVTGCACCACACTCCTGATGACJH6/1/20152060O.luridaQ9WV18
1660Flk_GRB2_FWDTCAGAACTGGTTCAAAGCTGAGTJH6/1/20152360O.luridaGRB2 FlankingP62994
1659Flk_GRB2_REVACTGCGCTGACATACTGGACJH6/1/20152060O.luridaP62994
1658Flk_H3.3_FWDCCAATGACAAATGAGCCACACAAJH6/1/20152360O.luridaH3.3 FlankingQ6P823
1657Flk_H3.3_REVTCGTACAAAGCAAACTGCACGJH6/1/20152160O.luridaQ6P823
1656Flk_H2A.V_FWDGCGATGGAGTTGATGAGGTGJH6/1/20152059O.luridaH2A.V FlankingP08991
1655Flk_H2A.V_REVCAAGGCAGTTTCTCGTTCGGJH6/1/20152059O.luridaP08991
1654Flk_H2A_FWDTGCTGGGGTTTTTCTGGGTCJH6/1/20152060O.luridaH2A FlankingP02270
1653Flk_H2A_REVTCAGGACGTGGTAAAGGAGGAJH6/1/20152160O.luridaP02270
1652Flk_p29ING_FWDGTGGACACACATGCACTCCTJH6/1/20152060O.luridap29ING4 FlankingQ8C0D7
1651Flk_p29ING_REVAAGCAGACTCAGATTCAGGCJH6/1/20152058O.luridaQ8C0D7

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

Reagent Table

Reaction_ComponentsVolume (ul)Final Concentration
2x Apex Red12.5125
Forward Primer (10uM)0.55
Reverse Primer (10uM)0.55
H2010.5105
1:2 cDNA1
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:

TempTime
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
ReagentVolume
1X Low TAE175-200 ml
Agarose2.3-2.6 g
EtBr17.5-20 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-SCARMBMP2
LadderNT1HT1ST1NC1HC1SC1NTCLadderNT1HT1ST1NC1HC1SC1NTCLadderNT1HT1ST1
BMP2HSPb11PGE/EP4
NC1HC1SC1NTCLadderNT1HT1ST1NC1HC1SC1NTCLadderNT1HT1ST1NC1HC1SC1NTC


Gel 2 layout:
GABABRGRB2H3.3
LadderNT1HT1ST1NC1HC1SC1NTCLadderNT1HT1ST1NC1HC1SC1NTCLadderNT1HT1ST1
H3.3H2A.VH2A
NC1HC1SC1NTCLadderNT1HT1ST1NC1HC1SC1NTCLadderNT1HT1ST1NC1HC1SC1NTC
p291N4
LadderNT1HT1ST1NC1HC1SC1NTCLadderEmptyEmptyEmptyEmptyEmptyEmptyEmptyEmptyEmptyEmptyEmpty


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. 
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