Ran a PCR to obtain luciferase DNA for sequencing.
Used sea pen gDNA extracted by Jonathan on 20150527.
Master mix calcs are here: 20150702_seapen_PCR
- 95C – 10mins
- 95C – 15s
- 55C – 15s
- 72C – 1min
- Go to Step 2 39 times
Ran samples on 0.8% agarose, low TAE gel stained with EtBr.
Lane 1 – ladder
Lane 2 – empty
Lane 3 – sea pen gDNA
Lane 4 – NTC
PCR did not work. Was expecting a band of ~800bp.
Looks like I may have overloaded the PCR reaction with gDNA. Used 10μL of gDNA.
However, that is quite the smear, suggesting a significant amount of degradation present in the gDNA.
Will re-run this PCR next week with less gDNA (or, cDNA instead) in order to generate a PCR product.
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.
Purified DNA from the remaining PCR bands excised by Jake on 20150609 and 20150610, as well as Jonathan’s sea pen PCRs from 20150604, using Ultrafree-DA spin columns (Millipore). Transferred gel pieces from storage tubes (1.5mL snap cap tubes) to spin columns. Spun 10,000g, 5mins @ RT. Transferred purified DNA back to original storage tubes. See the sequence_log (Google Sheet) for a full list of the samples and the sequencing plates layouts. Purified DNA was stored @ 4C O/N. Will prepare and submit plates for Sanger sequencing tomorrow.