Samples were submitted for sequencing to the University of Washington HTGU, two times from each direction using vector M13F/R primers. See Sequence Log for plate layout.
Selected 10 colonies (1-8, 18, 28) for mini-preps. Inoculated 5mL 1x LB + 50ug/mL of Kanamycin. Incubated O/N, 37C, 200RPM. 3mL of each culture were used for mini-preps. Used Qiagen kit. Samples were eluted w/30uL of EB.
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.
Will select 10 colonies for mini-preps.
Cloned the purified “qPCR Fragment” from 20111006 using the TOPO TA Cloning Kit (Invitrogen). Performed a half reaction (total volume = 3uL), using 1uL of purified PCR product. Incubated at RT for 20mins and then placed reaction on ice. Transformed chemically competent TOP 10 cells (Invitrogen) and heat shocked at 42C for 30s. Added 250uL of RT S.O.C. medium and incubated at 37C, 200RPM. Plated cells on pre-warmed Kan50+X-Gal plates (plates from 20110726; X-Gal added ~30mins before plating cells). Incubated plates O/N, 37C.
Good number of white colonies (>30). Will screen each colony with both qPCR primer sets to see if we can differentiate between the two COX/PGS isoforms in these clones.
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.
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.
Inoculated 4 x 5mL 1xLB + Kan50 with a colony from each set of clones, incubated 37C, 200RPM, O/N. Will mini prep and send for sequencing tomorrow.
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.