Jake Heare Research Central: qPCR

Showing posts with label qPCR. Show all posts

Wednesday, July 15, 2015

7 14 2015 Elongation qPCR run 2

In an effort to find a better normalizing gene than actin I searched our primer database for an elongation factor for olys. I found one and have run a pair of qPCR on them. This is the second of the runs.You can see the first here.

Primers:

1509	Ol_Ef1a_F	TCCTTGTTCAAACCCTTCATGT	BC	6/13/2012	22	40.9	58.1	O.lurida	Elongation factor 1-alpha
1508	Ol_Ef1a_R	CAAACCCTTGCCTCGGTAAG	BC	6/13/2012	20	55	58.8	O.lurida	Elongation factor 1-alpha

Reagent Table:

	Volume	Reactions X116
Ssofast Evagreen MM	10	1160
FWD Primer	0.5	58
REV Primer	0.5	58
1:9 cDNA	9

Added reagents from greatest to least volume
Vortexed
Centrifuged briefly
Pipetted 11 ul Master Mix to each tube
Pipetted 9 ul of 1:9 cDNA each column using a channel pipetter
Centrifuged plate at 2000 rpm for 1 minute
Ran Program Below

Program:

Step	Temperature	Time
Initiation	95 C	10 min
Elongation	95 C	30 sec
	60 C	1 min
Read
	72 C	30 sec
Read
Repeat Elongation 39 times
Termination	95 C	1 min
	55 C	1 sec
Melt Curve Manual ramp 0.2C per sec Read 0.5 C	55 - 95 C	30 sec
	21 C	10 min
End

Plate Layout:

1	2	3	4	5	6	7
DNased 42215 HC1	DNased 42215 NC1	DNased 42215 SC1	DNased 42215 HT1 1	DNased 42215 NT1 1	DNased 42215 ST1 1	NTC
DNased 42215 HC2	DNased 42215 NC2	DNased 42215 SC2	DNased 42215 HT1 2	DNased 42215 NT1 2	DNased 42215 ST1 2	NTC
DNased 42215 HC3	DNased 42215 NC3	DNased 42215 SC3	DNased 42215 HT1 3	DNased 42215 NT1 3	DNased 42215 ST1 3	NTC
DNased 42215 HC4	DNased 42215 NC4	DNased 42215 SC4	DNased 42215 HT1 4	DNased 42215 NT1 4	DNased 42215 ST1 4	NTC
DNased 42215 HC5	DNased 42215 NC5	DNased 42215 SC5	DNased 42215 HT1 5	DNased 42215 NT1 5	DNased 42215 ST1 5
DNased 42215 HC6	DNased 42215 NC6	DNased 42215 SC6	DNased 42215 HT1 6	DNased 42215 NT1 6	DNased 42215 ST1 6
DNased 42215 HC7	DNased 42215 NC7	DNased 42215 SC7	DNased 42215 HT1 7	DNased 42215 NT1 7	DNased 42215 ST1 7
DNased 42215 HC8	DNased 42215 NC8	DNased 42215 SC8	DNased 42215 HT1 8	DNased 42215 NT1 8	DNased 42215 ST1 8

Results:

All samples

NTCs

This like yesterday run also have very little amplification. Only one sample showed amplification of the target. Overall this is not a good gene for normalization. Today I'm going to run through my histone targets to see if they work as normalization genes.

You can see the raw data here.

Tuesday, July 14, 2015

7 14 2015 Elongation qpcr run 1

In an effort to find a better normalizing gene than actin I searched our primer database for an elongation factor for olys. I found one and have run a pair of qPCR on them. This is the first of the runs.

Primers:

1509	Ol_Ef1a_F	TCCTTGTTCAAACCCTTCATGT	BC	6/13/2012	22	40.9	58.1	O.lurida	Elongation factor 1-alpha
1508	Ol_Ef1a_R	CAAACCCTTGCCTCGGTAAG	BC	6/13/2012	20	55	58.8	O.lurida	Elongation factor 1-alpha

Reagent Table:

	Volume	Reactions X116
Ssofast Evagreen MM	10	1160
FWD Primer	0.5	58
REV Primer	0.5	58
1:9 cDNA	9

Added reagents from greatest to least volume
Vortexed
Centrifuged briefly
Pipetted 11 ul Master Mix to each tube
Pipetted 9 ul of 1:9 cDNA each column using a channel pipetter
Centrifuged plate at 2000 rpm for 1 minute
Ran Program Below

Program:

Step	Temperature	Time
Initiation	95 C	10 min
Elongation	95 C	30 sec
	60 C	1 min
Read
	72 C	30 sec
Read
Repeat Elongation 39 times
Termination	95 C	1 min
	55 C	1 sec
Melt Curve Manual ramp 0.2C per sec Read 0.5 C	55 - 95 C	30 sec
	21 C	10 min
End

Plate Layout:

1	2	3	4	5	6	7
DNased 42215 HC1	DNased 42215 NC1	DNased 42215 SC1	DNased 42215 HT1 1	DNased 42215 NT1 1	DNased 42215 ST1 1	NTC
DNased 42215 HC2	DNased 42215 NC2	DNased 42215 SC2	DNased 42215 HT1 2	DNased 42215 NT1 2	DNased 42215 ST1 2	NTC
DNased 42215 HC3	DNased 42215 NC3	DNased 42215 SC3	DNased 42215 HT1 3	DNased 42215 NT1 3	DNased 42215 ST1 3	NTC
DNased 42215 HC4	DNased 42215 NC4	DNased 42215 SC4	DNased 42215 HT1 4	DNased 42215 NT1 4	DNased 42215 ST1 4	NTC
DNased 42215 HC5	DNased 42215 NC5	DNased 42215 SC5	DNased 42215 HT1 5	DNased 42215 NT1 5	DNased 42215 ST1 5
DNased 42215 HC6	DNased 42215 NC6	DNased 42215 SC6	DNased 42215 HT1 6	DNased 42215 NT1 6	DNased 42215 ST1 6
DNased 42215 HC7	DNased 42215 NC7	DNased 42215 SC7	DNased 42215 HT1 7	DNased 42215 NT1 7	DNased 42215 ST1 7
DNased 42215 HC8	DNased 42215 NC8	DNased 42215 SC8	DNased 42215 HT1 8	DNased 42215 NT1 8	DNased 42215 ST1 8

Results:

All samples

NTCs

It appears that this run failed hard. There was no strong amplification in the samples. The NTCs had no amplification either. I only saw this data after I started the second replicate. I don't expect it to do much better. I think a trial with H3.3, H2A, and H2A.V could show if they would be good normalizing genes. I'll run them tomorrow. I'll post about the second elongation run when it finishes.

You can see the raw data here.

7 13 2015 TLR replicate rerun 2

Today I ran TLR2.1 using the optimized qPCR protocol. I wanna double check this target one last time before moving on to other genes. This is the second of the two plates I ran on TLR2.1, you can see the first here.

Primers:

1630	TLR2.1_FWD	ACAAAGATTCCACCCGGCAA	JH	5/21/2015	20	55		O.lurida	Toll-like receptor 2 type-1	Q9DD78
1629	TLR2.1_REV	ACACCAACGACAGGAAGTGG	JH	5/21/2015	20	55		O.lurida	Toll-like receptor 2 type-1	Q9DD78

Reagent Table:

	Volume	Reactions X116
Ssofast Evagreen MM	10	1160
FWD Primer	0.5	58
REV Primer	0.5	58
1:9 cDNA	9

Added reagents from greatest to least volume
Vortexed
Centrifuged briefly
Pipetted 11 ul Master Mix to each tube
Pipetted 9 ul of 1:9 cDNA each column using a channel pipetter
Centrifuged plate at 2000 rpm for 1 minute
Ran Program Below

Program:

Step	Temperature	Time
Initiation	95 C	10 min
Elongation	95 C	30 sec
	60 C	1 min
Read
	72 C	30 sec
Read
Repeat Elongation 39 times
Termination	95 C	1 min
	55 C	1 sec
Melt Curve Manual ramp 0.2C per sec Read 0.5 C	55 - 95 C	30 sec
	21 C	10 min
End

Plate Layout:

1	2	3	4	5	6	7
DNased 42215 HC1	DNased 42215 NC1	DNased 42215 SC1	DNased 42215 HT1 1	DNased 42215 NT1 1	DNased 42215 ST1 1	NTC
DNased 42215 HC2	DNased 42215 NC2	DNased 42215 SC2	DNased 42215 HT1 2	DNased 42215 NT1 2	DNased 42215 ST1 2	NTC
DNased 42215 HC3	DNased 42215 NC3	DNased 42215 SC3	DNased 42215 HT1 3	DNased 42215 NT1 3	DNased 42215 ST1 3	NTC
DNased 42215 HC4	DNased 42215 NC4	DNased 42215 SC4	DNased 42215 HT1 4	DNased 42215 NT1 4	DNased 42215 ST1 4	NTC
DNased 42215 HC5	DNased 42215 NC5	DNased 42215 SC5	DNased 42215 HT1 5	DNased 42215 NT1 5	DNased 42215 ST1 5
DNased 42215 HC6	DNased 42215 NC6	DNased 42215 SC6	DNased 42215 HT1 6	DNased 42215 NT1 6	DNased 42215 ST1 6
DNased 42215 HC7	DNased 42215 NC7	DNased 42215 SC7	DNased 42215 HT1 7	DNased 42215 NT1 7	DNased 42215 ST1 7
DNased 42215 HC8	DNased 42215 NC8	DNased 42215 SC8	DNased 42215 HT1 8	DNased 42215 NT1 8	DNased 42215 ST1 8

Results:

All samples

NTCs

There was some minor amplification in two of the 4 NTCs but it appears to be a product much larger than the target. This replicate looks much better than the previously one. I'm going to run some scripts on the raw fluoroescence to determine how the replicates compare to each other.

You can see the raw data file here.