Tag Archives: cDNA

PCR – Full-length PGS1 & PGS2 cDNAs

Ran PCR to amplify full-length cDNAs of PGS1 & PGS2 (COX1 & COX2) using primers designed to anneal in the 5’/3’UTRs of each isoform. PGS1 primers = SRIDs: 1377, 1378. PGS2 primers = 1376, 1375. Master mix calcs and cycling params are here. cDNA was pooled cDNA made 20110311 from various tissues.

PGS1 Expected Size = ~2300bp

PGS2 Expected Size = ~2500bp

Results:

Gel

Lane 1 – Hyperladder I (Bioline)

Lane 2 – PGS1

Lane 3 – PGS1 NTC

Lane 4 – PGS1 NTC

Lane 5 – PGS2

Lane 6 – PGS2 NTC

Lane 7 – PGS2 NTC

PGS1 Results: PGS1 PCR produces a single band of the expected size (~2300bp), indicating that the two primers, which were designed to anneal in the 5’/3’UTRs of the gene and should be highly specific to just this isoform, work perfectly. The band was excised and stored @ -20C in “Sam’s Miscellaneous” box.

PGS2 Results: PGS2 PCR didn’t produce any product. Will repeat with a lower annealing temp (50C instead of 55C).

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qPCR – C.gigas V.vulnificus Exposure cDNA (from 20110311)

Ran a qPCR using 3hr Vibrio vulnificus exposure cDNA from 20110311. Original experiment conducted on 20110111 with defensin primers (SR IDs: 1109 & 1070) and GAPDH (SR IDs: 1172 & 1173). Master mix calcs are here. Cycling params, plate layout, etc can be seen in the qPCR Report (see Results). This was performed to help Herschel.

Results:

qPCR Report (PDF)

qPCR Data File (CFX96)

Initial glance at data looks good. GAPDH exhibits highly consistent Cq values across all samples, controls and exposed. Although, there is slight amplification of something in the two water samples for GAPDH, the melt curve shows that this product has a different melting temperature than our intended target. As such, I believe the GAPDH data to be useable, since no other samples exhibit this smaller product. Defensin shows clean water sample and clean melt curves with a single peak. However, it seems like we may not see an effect on defensin expression in response to the Vibrio vulnificus exposure…

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5’/3′ RACE – C.gigas COX2/PGS2 Nested RACE PCR

Performed nested RACE PCR on the RACE PCR products generated on 20110722 using the following nested primers: PGS2_ngspRACE_5′ (SR ID: 1350) and PGS2_ngspRACE_3′ (SR ID: 1349). Removed 2uL from each of the primary PCR reactions and brought up to 100uL in tricine EDTA (supplied in the Clontech SMARTer RACE cDNA Amplification Kit). Performed the nested RACE PCR according to the Clontech manual. Briefly, this is the same as the primary RACE PCR reaction, but using 5uL of the diluted primary PCR product and 1uL of the Nested Universal Primer (instead of 5uL of the 10X Universal Primer Mix). Master mix calcs and set up are here. Cycling params followed “Program 2″ of the Clontech protocol, modified for nested primers, and are as follows:

20 cycles:

94C 30s

68C 30s

72C 3m

Results:

Gel Layout:

Lane 1 – Hyperladder 1

Lanes 2-6 = 5′ RACE Library

Lane 2 – nGSP1 (5′ RACE primer)

Lane 3 – nGSP2 (3′ RACE primer)

Lane 4 – Neg. Control (no RACE primers)

Lane 5 – Neg. Control (nGSP1, no Universal primer)

Lane 6 – Neg. Control (nGSP2, no Universal primer)

Lane 7 – Empty

Lanes 8-12 = 3′ RACE Library

Lane 8 – nGSP1 (5′ RACE primer)

Lane 9 – nGSP2 (3′ RACE primer)

Lane 10 – Neg. Control (no RACE primers)

Lane 11 – Neg. Control (nGSP1, no Universal primer)

Lane 12 – Neg. Control (nGSP2, no Universal primer)

First of all, we see the appropriate response of each primer only producing amplicons in their respective libraries (i.e. 5′ primer only works in 5′ RACE library). This simply confirms that the primers were designed correctly. Secondly, our negative controls are clean. Thirdly, we get distinct bands from both primers. The bands marked with blue arrows in the image above were excised and purified using Ultrafree DA spin columns (Millipore). These products will be used for cloning and eventual sequencing.

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qPCR – C.gigas GAPDH second rep on V.vulnificus exposure cDNA (from 20110311) and standard curves for COX1, COX2, GAPDH

Ran a qPCR on all cDNA samples. Created a standard curve to possibly allow for use of the BioRad software for gene expression analysis. Standard curve was created from pooled cDNA (1uL from each individual sample). Master mix calcs are here.

Results:

qPCR Data File (BioRad CFX96)

qPCR Report (PDF)

Standard curves aren’t that good. Will not use them. Will analyze data using PCR Miner.

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qPCR – C.gigas actin and GAPDH on V.vulnificus exposure cDNA (from 20110311)

Ran a qPCR on all cDNA samples from the V.vulnificus exposure experiment from 20110111. This qPCR was to test 2 of 4 potential normalizing genes to evaluate which genes show the least amount of effect from the treatments in this experiment. Primers for actin used were Cg_Actin_306_F (SR ID: 1170), Cg_Actin_408_R (SR ID: 1171). Samples were run in duplicate. Master mix calcs are here. The master mix info is the same that was used earlier today, but with the primers noted above, not those listed on the calcs page. Plate layout, cycling params, etc., can be seen in the qPCR Report (see Results).

Results:

qPCR Data File (BioRad CFX96)

qPCR Report (PDF)

Actin: Average Cq = 20.21, Standard Deviation = 1.22

GAPDH: Average Cq = 24.42, Standard Deviation = 0.519

Based on the results from the 4 normalizing genes examined, I will use GAPDH as the normalizing gene due to it having the lowest standard deviation of the 4 normalizing genes. Will perform another qPCR to run a duplicate of GAPDH so that we have a second rep.

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qPCR – C.gigas 18s and EF1a on V.vulnificus exposure cDNA (from 20110311)

Ran a qPCR on all cDNA samples from the V.vulnificus exposure experiment from 20110111. This qPCR was to test 2 of 4 potential normalizing genes to evaluate which genes show the least amount of effect from the treatments in this experiment. Primers for 18s used were Cg_18s_1644_F (SR ID: 1168), Cg_18s_1750_R (SR ID: 1169). Primers for EF1a used were EF1_qPCR_5′ (SR ID: 309), EF1_qPCR_3′ (SR ID: 310)Samples were run in duplicate. Master mix calcs are here. The master mix info is the same that was used earlier today, but with the primers noted above, not those listed on the calcs page. Plate layout, cycling params, etc., can be seen in the qPCR Report (see Results).

Results:

qPCR Data File (BioRad CFX96)

qPCR Report (PDF)

18s: Average Cq = 22.39, Standard Deviation = 0.905

EF1a: Average Cq = 20.59, Standard Deviation = 0.658

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qPCR – C.gigas COX2 on V.vulnificus exposure cDNA (from 20110311)

Ran a qPCR on all cDNA samples from the V.vulnificus exposure experiment from 20110111. Primers used were Cg_COX1/2_qPCR_F (SR ID: 1192) & Cg_COX2_454align1_R (SR ID: 1190). Samples were run in duplicate. Master mix calcs are here. The master mix info is the same that was used earlier today, but with the primers noted above, not those listed on the calcs page. Plate layout, cycling params, etc., can be seen in the qPCR Report (see Results).

Results:

qPCR Data File (BioRad CFX96)

qPCR Report (PDF)

Data looks good (e.g. the replicates are all very close, with the largest Cq Std. Deviation = 1.227, although this does appear to be an anomaly as the next highest Cq Std. Deviation in any of the reps is 0.633), nothing in the NTCs, & the melt curves look good. Will eventually normalize the data and then perform a complete analysis.

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qPCR – C.gigas COX1 on V.vulnificus exposure cDNA (from 20110311)

Ran a qPCR on all cDNA samples from the V.vulnificus exposure experiment from 20110111. Primers used were Cg_COX1/2_qPCR_F (SR ID: 1192) & Cg_COX1_qPCR_R (SR ID: 1191). Samples were run in duplicate. Master mix calcs are here. Plate layout, cycling params, etc., can be seen in the qPCR Report (see Results).

Results:

qPCR Data File (BioRad CFX96)

qPCR Report (PDF)

Data looks good (e.g. the replicates are all very close, with the largest Cq Std. Deviation = 0.534), nothing in the NTCs, & the melt curves look good. Will eventually normalize the data and then perform a complete analysis.

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qPCR – Hard Clam NGS Primer Checks

Ran a qPCR to evaluate a large batch of primers (40 sets) that were ordered per Steven, based off of the most recent SOLiD run (samples submitted 3/10/2011; see Dave’s notebook for more info). Pooled cDNA (2uL from each individual; from 20110511) was used. Master mix calcs are here. Plate layout, cycling params, etc. can be found in the qPCR Report (see Results). The list of primers tested is available in the Primer Database and consist of SR IDs 1233 – 1312. For brevity, samples were only labelled with the corresponding contig number.

Results:

qPCR Data File (BioRad CFX96)

qPCR Report (PDF)

Samples that produced good melt curves are listed here.

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qPCR – Lexie’s QPX Temp & Tissue Experiment (see Lexies Notebook 4/26/2011)

Ran qPCR with Lexie’s cDNA samples from this experiment with the following primer sets in order to better evaluate her biological reps:

QPX_SPB_F/R (SR ID: 387, 388)

LABY_A/Y (SR ID: 116, 121)

LABY was run as a potential normalizing gene. Master mix calcs are here. Plate layout, cycling params, etc. can be found in the qPCR Report (see Results). Samples were run in duplicate and were labeled according to what was written on the tops of Lexie’s cDNA tubes.

Results:

qPCR Data File (BioRad CFX96)

qPCR Report (PDF)

LABY primers worked, but the melt curves don’t look that good. I’ll let Lexie worry about the rest of the analysis.

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