Performed PCR on 40 colonies using both qPCR primer sets to see if I could differentiate between which colonies potentially contained each isoform to reduce the amount of clones needed for sequencing. Master mix and cycling params are here. Primers used were:
Cg_COX1/2_qPCR_F (SR ID: 1192)
Cg_COX1_qPCR_R (SR ID: 1191)
Cg_COX2_454align1_R (SR ID: 1190)
Positive controls for both primers set were also run. The positive control template was the purified PCR product from 20111006.
Ladder is Hyperladder II (Bioline). Samples are loaded, left to right, as PGS1 and PGS2 on each colony (e.g. on the bottom gel image, under the “Colony 40″ label is the PGS1 rxn on the left and the PGS2 rxn on the right).
Nearly every colony exhibits amplification using both primer sets, w/the PGS1 reaction producing a band of ~250bp and the PGS2 reaction producing a band of ~750bp. Colonies 18 and 28 are an exception to this and produced no band with the PGS2 primer set. NTCs were clean. The positive controls worked as expected, yielding a band of ~250bp for PGS1 and a band of ~250bp for PGS2.
It is confusing as to why the size of the PGS2 positive control is different than the product that was generated from the colony PCRs.
Ran PCR using primers Cg_COX1/2_qPCR_F, Cg_COX1_qPCR_R, Cg_COX2_454align1_R (SR IDs: 1192, 1191, 1190; respectively). Template was pooled cDNA from 20110311 of various C.gigas tissues. These reactions will verify (sort of) if we have both isoforms present in the PCR performed earlier today, prior to cloning. Master mix calcs and cycling params are here.
Lane 1: Hyperladder I (Bioline)
Lane 2: COX1/PGS1 primer set
Lane 3: COX1/PGS1 primer set NTC
Lane 4: COX2/PGS2 primer set
Lane 5: COX2/PGS2 primer set NTC
NTCs are clean for both primer sets. We see bands of the expected size for both primer sets. Additionally, we see lower expression in COX2/PGS2, as we observed in our previous qPCR reactions with these primer sets. Will clone the large fragment that was PCR’ed/purified from earlier today.
Ran PCR using primers Cg_COX_982_F and Cg_COX_2138_R (SR IDs: 1149 & 1151, respectively). Template was pooled cDNA from 20110311 of various C.gigas tissues. These primers anneal 5′ and 3′ of where the qPCR primers for both COX1/PGS1 and COX2/PGS2 anneal. Master mix calcs and cycling params are here. Ran multiples of the same reaction to ensure sufficient product for use in cloning/PCR.
Gel is loaded with Hyperladder I (Bioline) and 7 samples (no NTC; don’t ask). Band in each lane is of the expected size (~1200bp). Each band was excised and purified using Ultra-free DA columns (Millipore), according to protocol. Purified DNA will be used in a subsequent PCR using the qPCR primers for COX/PGS 1&2 BEFORE cloning this product for sequencing.
Repeated PCR from 20110825 to attempt to amplify the full-length cDNA for PGS2 (COX2), however this time using a more robust polymerase (Amplitaq Gold) in hopes of getting results. Additionally, tried 3 different Mg2+ concentrations (1.5mM, 2.0mM, and 3.0mM). Master mix calcs and cycling params are here. cDNA was pooled cDNA made 20110311 from various tissues. PGS2 primers = 1376, 1375.
PGS2 expected size = ~2500bp
Loading order doesn’t matter, as there are no bands. Ladder is Hyperladder I (Bioline). Will continue current sequence analysis and potentially design a new set of primers…
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
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).
Used new primers for sequencing (SR IDs: 1351 & 1352) clone #4. Sequenced clone two times in each direction. DNA and primers were sent for sequencing at ASU. Requested “High GC” treatment to help overcome the issue seen on 20110728.
Sequencing results received 20110810. Initial analysis suggests that we managed to fully sequence this clone! Will try to assemble a full-length CDS for COX2/PGS2.
Isolated plasmid DNA from 3mL of liquid cultures that were inoculated yesterday using Qiagen’s miniprep kit. DNA was eluted with 50uL of EB. DNA was prepped and sent for sequencing to ASU sequencing facility. Each clone was sequenced two times in each direction. Samples are as follows:
Name – Clone # Primer
SJW01 – 1 M13F
SJW02 – 1 M13F
SJW03 – 1 M13R
SJW04 – 1 M13R
SJW05 – 2 M13F
SJW06 – 2 M13F
SJW07 – 2 M13R
SJW08 – 2 M13R
SJW09 – 3 M13F
SJW10 – 3 M13F
SJW11 – 3 M13R
SJW12 – 3 M13R
SJW13 – 4 M13F
SJW14 – 4 M13F
SJW15 – 4 M13R
SJW16 – 4 M13R
Clone #s are as follows:
1 – 5′ Library Top band
2 – 5′ Library Mid band
3 – 5′ Library Bottom band
4 – 3′ Library band
Sequencing results received 20110801. SJW15 and 16 apparently stop abruptly. The sequencing facility believes this to be caused by secondary structure of the template. Depending on how things align, I may consider using 7-daeza-GTP in a PCR reaction and re-sequencing this clone, as the 7-daeza-GTP helps relax secondary structure.
Spoke with Steven and he suggested just designing new primers closer to each other and resubmit.
Performed colony PCRs on the 4 sets of cloning reactions that were performed yesterday using the M13F/R vector primers. Colonies were picked, restreaked on a fresh LB Kan50 plates (made 20110726 by SJW) and PCR’d. Master mix calcs are here. Selected 8 white colonies from each cloning reaction for PCR. Restreaked plate was incubated @ 37C O/N.
95C – 10m
95C – 10s
55C – 10s
72C – 3m
Hyperladder I is used as the ladder in both gels.
Cloning results look great (except Colony #1 in the 5′ Top Band didn’t produce a product). Will select a re-streaked colony from each set and inoculate liquid culture for mini prep and subsequent sequencing.
The bands that were excised and purified earlier today were cloned in to pCR2.1 using the TOPO TA Cloning Kit (Invitrogen) according to the manufacturer’s protocol with the following changes: used 4uL of all PCR products, incubated ligation reactions for 15mins @ RT, incubated competent cells with ligation reactions for 15mins on ice.
Two volumes of each reaction were plated (50uL and 100uL) on Kan50 plates with X-gal (made 20010412 by SJW) and incubated @ 37C O/N.
Ample number of white colonies for all 4 sets of cloning reactions.