Each clone’s sequence matches that of the source sequence, so we’re good to go!
Will proceed with dye-based quantification of each plasmid. Will then proceed with developing ISH probes (membrane genes 1 & 2, tail fiber gene) or qPCR standard curves (DNA helicase, head-to-tail).
In the alignments below, the reference sequence is highlighted in light yellow. The two electropherograms are align below the reference. The grey line in the consensus sequence indicates any sequence disagreements by placement of a black mark at the position. However, the sequences all match, so there are no black marks in the regions between the identified vector sequences (red annotations below each electropherogram).
Submitted ~500ng of each plasmid in a final volume of 15μL (including primer). Each clone will be sequenced from each direction with M13F (-21) (25pmol; 2.5μL of 10μM stock) and M13R primers (25pmol; 2.5μL of 10μM stock) for a total of 10 sequencing reactions:
Prepared two DNA plates and corresponding primer plates for sequencing at the UW HTGC from the purified gel-purified PCRs from yesterday. Primer plates were prepared by adding 7μL of NanoPure H2O to each well and then adding 3μL of 10μM primer to the appropriate wells. For the DNA plates, added 10μL of DNA to the appropriate wells.
NOTE: The H2A_ST1 samples had insufficient volume of DNA for all four sequencing reactions. Added 30μL of NanoPure water to purified DNA, mixed and distributed to the appropriate wells.
Sequencing plates layouts can be seen here (Google Sheet): sequence_log.
Submitted the plates to the UW HTGC for Sanger 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.
Sent plasmid prep to ASU (5uL of plasmid + 1uL of 10uM M13F/R). SJW01 = M13F, SJW02 = M13R.
Sequencing looks great! Definitely have a portion of the second isoform of COX/PGS!! Here’s the result of the consensus BLASTed in GenBank>Nucleotide (others)>blastn:
Top hit in the db is COX1/PGS1, and, clearly, there are differences between the two sequences confirming that we have the second isoform (COX2/PGS2). Will design more RACE primers in hopes of obtaining the full-length cDNA sequence.