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.
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.
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
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.
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.
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)
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.
Performed reverse transcription on DNased RNA from the hard clam vibrio tubiashii challenge experiment (see Dave’s Notebook 5/2/2011), following the Promega M-MLV RT protocol with ~1ug of DNAsed RNA. Master mix calcs are here. Reactions were done in a plate. cDNA was diluted 1:4 with H2O.
Due to the failure of the primary PCR on both 5′ and 3′ RACE cDNA libraries (from 20080619) yesterday, will perform nested PCR using a nested GSP designed by Steven (CgPGSRACEsrNGSP1; SR ID:1209). The 5uL of PCR reactions that were set aside yesterday were diluted to 250uL with tricine-EDTA (supplied with the Clontech SMARTer RACE cDNA Amplification Kit) as instructed in the Clontech manual. The master mix and tube layouts were exactly the same as yesterday’s, but instead of using 2.5uL of RACE cDNA library as template, I used 5uL of the diluted PCR reaction. Additionally, Universal Nested Primers were used instead of the Universal Primer Mix (both supplied in the kit). Cycling parameters followed “Program 2″ from the Clontech manual for 25 cycles.
Entire PCR rxns were run on a 1.2% gel, as instructed by the Clontech manual.
Results:
So….. What we see here is a melted gel!
But! We also see a successful PCR! The first three lanes (excluding the Hyperladder I) are 5′ RACE rxn, followed by two different negative controls (the negative control in the last lane is the one we’re really concerned with and it’s totally clean). The next three lanes are the 3′ RACE rxn, followed by two different negative controls (the negative control in the last lane is the one we’re really concerned with and it’s totally clean). As hoped/expected, we got a nice, clear product in the 5′ RACE rxn.
The bright band (~1500bp) in the 5′ RACE rxn PCR was excised and purified using Millipore Ultrafree DA columns according to protocol. Will clone and sequence this product.
Performed qPCR using cDNA from 20110311. This was performed for 2 reps with TIMP3(BB) (SR IDs: 1067 & 1106). Master mix calcs are here. Plate layout, cycling params, etc. can be found in the qPCR Report (see Results).
Results:
qPCR Report (PDF)
qPCR Data File (CFX96)
