20110603
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.
20110602
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.
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
20110601
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.
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.
20110523
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 labeled with the corresponding contig number.
Results:
qPCR Data File (BioRad CFX96)
qPCR Report (PDF)
Samples that produced good melt curves are listed here.
20110520
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.
qPCR - Emma's New 3KDSqPCR Primers
Due to previous contamination issues with Emma's primers, Emma asked me to order new primers, reconstitute them and run a qPCR for her to see if we could eliminate her contamination issues with this primer set. cDNA template was supplied by Emma (from 2/2/11) and was from a C.gigas 3hr Vibrio vulnificus challenge. Samples were run in duplicate, as requested.
Master mix calcs are here. Plate layout, cycling params, etc. can be found in the qPCR Report (see Results). Primer set used was:
Cg_3KDSqPCR_F/R (SR IDs: 1186, 1187)
Results:
qPCR Data File (BioRad CFX96)
qPCR Report (PDF)
The negative controls (NTC) are negative, meaning they do not cross the threshold set by the BioRad software. However, there is clearly amplification in the NTCs, but they come up late enough that they do not cross the threshold and, thus, generate a Cq value. Additionally, the melt curve reveals peaks in the NTCs that are at the same melting temperature as the product produced in the cDNA qPCR reactions. This would potentially imply some sort of contamination, as Emma has experienced.
Honestly, I do no think contamination is the problem. I believe that the "contamination" being seen in the NTCs is actually primer dimer. Increasing the annealing temperature (I'm not sure if Emma tried this during her troubleshooting) could potentially alleviate this issue. However, I'm not sure she's amplifying the target that she wants to. Based on my analysis, I think she needs to re-design primers for her 3KDS target. Read my analysis and why I came to this conclusion below.
It seems unlikely that two independent people (and multiple primer stock replacements!) would have contamination, so I looked in to things a bit further.
I BLASTed the primer sets (NCBI, blastn, est_others db, C.gigas only) and the BLAST results reveal the primers matching with a C.gigas EST sequence that would produce a band of only 63bp. Here's a screen capture of the BLAST results:
This result does NOT agree with what is entered in our Primer Database. As entered in our sheet, the expected PCR product would be ~102bp. However, taking in to account the BLAST results, it would be difficult to distinguish the difference between primer dimers and PCR product in a melt curve analysis.
Emma has previously run a conventional PCR with these primers and ran a gel (see below). At the time, it was thought to be contamination, but in retrospect (knowing the results of the qPCRs and the BLAST results) it seems likely that what she's seeing in the negative controls was actually primer dimer, which was the same size of her PCR product (which she thought should be larger). Additionally, the gel was difficult to interpret because no ladder was run. A ladder might have revealed that her PCR product was half the size that she was expecting:
20110511
Reverse Transcription - Hard Clam Gill DNased RNA (from 20110509)
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.
20110509
DNase - Hard Clam Gill RNA (from earlier today)
DNased 5ug of RNA from each sample with Ambion's Turbo DNA-free Kit, according to the manufacturer's rigorous protocol. Samples were spec'd and stored @ -80C in the "Hard Clam V.t. Experiment RNA" box.
Results:
Due to lack of a positive control (and lack of primers known to amplify gDNA), these DNased RNA samples will NOT be checked to verify elimination of gDNA carryover at this time. Will proceed with making cDNA for qPCR anyway.
RNA Isolation - Hard Clam Gill Tissue from Vibrio Experiment (see Dave's Notebook 5/2/2011)
Isolated RNA in 1mL of Tri-Reagent according to the manufacturer's protocol. Also, finished RNA isolation of samples that were started 20110506. Samples were resuspended in 50uL 0.1%DEPC-H2O and spec'd.
Results:
20110508
Primer Design - Hard Clam NGS Primers
Designed primers for
40 PCR targets derived from the most recent SOLiD data by Steven(Evernote link) using
BatchPrimer3.
BatchPrimer3 results are here.
20110506
RNA Isolation - Hard Clam Gill Tissue from Vibrio Experiment (see Dave's Notebook 5/2/2011)
Isolated RNA in 1mL of Tri-Reagent according to manufacturer's protocol. Samples were precipitated with isopropanol and stored over the weekend @ -20C. Will conclude isolation on Monday. The samples isolated were:
MA 1-11
MA Vt 1-11
20110428
Received - Live Hard Clams From Scott Lindell
Received two bags containing ~24 live clams (didn't count) in each bag. One bag labeled as "Mashpee Control" and the other "BX Selected." Clams were stored @ 4C by Steven.
20110426
Bacterial Cultures - Colonies Selected from Yesterday's Colony PCRs
Inoculated 5mL of 1x LB + Kan50 (made by Steven on 3/23/11). Incubated O/N at 37C, 250RPM. Will perform mini preps tomorrow. The following samples were selected:
MM09 - #1, 2, 8
MM11 660bp - #1, 2, 8
MM10 2/8/11 - #1, 2
MM04 1/19/11 - #2, 3
MM11 3000bp - #3
MM04 1500bp - #4
MM06 1/19/11 - #1, 2
MM04 550bp - #1, 2
MM05 1/19/11 - #1, 2
Mini Preps - Liquid Cultures from yesterday
Mini prepped 3mLs of each culture, according to Qiagen protocol. Samples were eluted with 50uL of Buffer EB.
20110425
Bacterial Cultures - Colonies Selected by Steven from Steven's Re-Streaked Plate
Inoculated 5mL of 1x LB + Kan50 (made by Steven on 3/23/11). Incubated O/N @ 37C, 250RPM. Will perform mini preps tomorrow. The following samples were selected (red text on the plate):
#9
#10
#13
#43
#45
#49
#56
#58
Colony PCR - Colonies from COX1 Genomic Cloning (from 20110411)
Ran colony PCR on various colonies produced from cloning on 20110411. All colonies were picked, re-streaked on Kan50 plate(s) and PCR'd.
Master mix calcs are here. Cycling params:
95C - 10mins
40cycles of:
95C - 30s
55C - 30s
72C - 3mins
72C - 10mins
Results:
20110422
Colony PCR - 5'RACE Colony: COX2 (repeat of yesterday's PCR)
Repeated yesterday's PCR on the re-streaked colony in order to run the product on a gel with a more appropriate ladder. See yesterday's entry for all PCR info.
Results:
Lane 1: Hyperladder I
Lane 2: colony PCR
Lane 3: NTC
A band of nearly ~950bp is seen in the colony PCR, suggesting that the cloning reaction was successful. However, this does not match up with the expected size of ~1500bp seen on 20110407. Will sequence this regardless. Also, the gel on 20110407 may not have run properly (see image from that dat), which could possibly explain why we don't see the "expected" size band of ~1500bp? Will inoculate a liquid culture for mini prep for eventual sequencing.
20110421
Colony PCR - 5' RACE Colony: COX2
One white colony (marked with arrow in image linked) from the two plates from Steven's cloning (from yesterday) was picked, restreaked on a new Kan50 plate (no X-gal) and PCR'd.
Master Mix:
2x Apex Red Master Mix - 25uL
10uM M13 Forward - 1uL
10uM M13 Reverse - 1uL
H2O - 23
Added 25uL to each PCR tube.
Cycling Params:
95C - 10mins
40 cycles of:
95C - 30s
55C - 30s
72C - 2mins
72C - 10mins
Results:
Lane 1: Hyperladder IV
Lane 2: colony PCR
Lane 3: NTC
Turns out the Hyperladder IV (this gel was run in the Friedman Lab) only goes up to 1000bp. So, the band we see in the colony PCR reaction could be close to the expected size if the insert is present (~1500bp). Although, we also see a band in the NTC. Will repeat this PCR and run on a gel with a more appropriate ladder...
20110414
Colony PCR - 5' RACE Colonies
Two light blue colonies (there were no white colonies) were picked, restreaked on a new Kan50 plate (no X-gal) and PCR'd.
Master Mix:
2x Apex Red Master Mix - 37.5uL
10uM M13 Forward - 3uL
10uM M13 Reverse - 3uL
H2O - 46.5
Added 25uL to each PCR tube.
Cycling Params:
95C - 10mins
40 cycles of:
95C - 30s
55C - 30s
72C - 2mins
72C - 10mins
Results:
The two pale blue colonies do NOT contain our desired insert. Despite the presence of a larger, faint band (~950bp), that is far smaller than our 5'RACE insert size (~1500bp). And, clearly, the primary amplicon produced is ~250bp, which is the expected size for empty vector... Ladder is Hyperladder I (Bioline).
Will need to re-do ligation reaction and will do so at the recommended volumes in the TOP TA (Invitrogen) protocol.
20110413
Additional Bacteria Plating - Remaining transformations from 20110411
Plated the remaining 200uL of transformed bacteria from the three reactions that produced no white colonies ( ) on Kan50 plates + X-gal. Incubated O/N 37C.
Results:
20110411
Ligations - COX1/COX2 PCR Products
Performed ligations/cloning on a variety of COX1 genomic and COX 5' RACE products using the TOPO TA Cloning Kit (Invitrogen). Used 2uL of gel-purified PCR product in each cloning rxn, 2.5uL of H2O, 1uL of salt solution, and 0.5uL of the Invitrogen pCR2.1 vector. Incubated samples for 5mins at RT. Used 2uL of the cloning reaction to transform TOP10 chemically competent cells (Invitrogen), mixed very gently, incubated on ice for 5mins, heat shocked at 42C for 30s and immediately placed on ice. Added 250uL of SOC Medium and incubated tubes at 37C, 200RPM for 1hr. Plated 50uL of each transformation on LB+Kan plates (made by Steven on unknown date with unknown Kan concentration) containing 40uL of 40mg/mL X-gal. Incubated O/N at 37C. Remaining volume of transformed bacteria were stored @ 4C.
Results:
20110407
5'/3' RACE PCRs - Nested PCRs for COX2 Sequence
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.
20110406
5'/3' RACE PCRs - COX2 Sequence on 5' & 3' RACE Libraries (from 20080619)
Ran PCRs on both the 5' & 3' RACE libraries created 20080619 with a new COX2 gene-specifc (GSP) primer designed by Steven (CgPGSRACEsrGSP1; SR ID: 1208). Although this primer was designed to obtain additional 5' sequence, it was used with both 5' and 3' libraries as a precaution in case it accidentally designed on the wrong strand. PCR rxn was set up according to the Clontech SMARTer RACE cDNA Amplification Kit.
Master mix calcs are here. PCR cycling followed "Program 1" from the Clontech manual for 25 cycles.
After PCR completion, 5uL were transferred to a clean tube and saved, in case this PCR didn't work and a nested PCR would need to be performed. This is according to the Clontech protocol. Samples were run on a 1.2% agarose gel, as instructed in the Clontech manual.
Results:
No bands of any kind in any sample, including the negative controls (gel not shown). Will perform nested PCR on both libraries in hopes of getting bands.