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

Wednesday, June 24, 2015

6 23 2015 TLR2.1 qPCR

Yesterday after discovering that the Opticon was acting up, I decided to run a couple qPCR's on the Biorad CFX in the Friedman lab. The first was the TLR2.1 gene which had not been present in the Dabob population during the initial primer checks.

Primer:
1630TLR2.1_FWDACAAAGATTCCACCCGGCAAJH5/21/20152055O.luridaToll-like receptor 2 type-1Q9DD78
1629TLR2.1_REVACACCAACGACAGGAAGTGGJH5/21/20152055O.luridaToll-like receptor 2 type-1Q9DD78

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
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:

All Samples


NTCs



So the graph doesn't really do justice for the results. There is amplification in a majority of samples but interestingly only about half of the Dabob population shows expression of this allele. The other half show absolutely no expression. There are a few individuals which show a very low expression of this allele but others show some of the highest expression. Overall this is pretty confusing and may be indicative that the Dabob population is more heterogeneous for this allele than the other populations. After running this qPCR, I ran another Actin qPCR using the friedman machine to create a normalizing standard which I will talk about in my next post.

You can view the raw data here.
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Tuesday, June 23, 2015

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:

1680Flk_HSP70_FWDGACCCGGATGTCCAGAGGAAJH6/9/20152060O.luridaHSP70 Flanking
1679Flk_HSP70_REVTCAGGACGGCTTGTGAACGJH6/9/20151960O.lurida
1678Flk_Actin_FWDTCGAGGGAGGAAGAGGAAGCJH6/9/20152059O.luridaActin Flanking
1677Flk_Actin_REVAACTGGGACGACATGGAGAAJH6/9/20152061O.lurida
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
1668Flk_BMP2_FWDGGCTGGCTGGATCGTCATJH6/1/20151860O.luridaBMP2 FlankingP12643
1667Flk_BMP2_REVATGGAGTCTGTGGACGGTTTGJH6/1/20152160O.luridaP12643

Reagent Table:
Reaction_ComponentsVolumeFinal Concentration
2x Apex Red12.5125
Forward Primer (10uM)0.55
Reverse Primer (10uM)0.55
H2010.5105
Template1
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 25 ul of each product on a 1.3% agarose gel

Gel Reagent Table
ReagentVolume
1X Low TAE175 ml
Agarose2.3 g
EtBr17.5 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:
ActinHSP70
LadderHC1NC1SC1HT1NT1ST1NTCLadderHC1NC1SC1HT1NT1ST1NTCEmptyEmptyEmptyEmpty
PGEEP4HSPb11
LadderHC1NC1SC1HT1NT1ST1NTCLadderHC1NC1SC1HT1NT1ST1NTCEmptyEmptyEmptyEmpty
BMP2
LadderHC1NC1SC1HT1NT1ST1NTCEmptyEmptyEmptyEmptyEmptyEmptyEmptyEmptyEmptyEmptyEmptyEmpty

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.  
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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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