Research+Page-+AlexH

=**Fall 2012**=

Alex, show your most recent work - Dr. B 11/19/12

Week 8 [1 kb ladder, 1°, 2°] ==> PCR^2 gel showed multiple bands and is definitely contaminated. By far, the most prominent band is much too small to be the desired product. The gel does show a light band at the size of the desired product, however there are many other bands and a general smear through the lane. Cloning cannot be continued from this PCR^2. ==> Literature shows that the leading 73 residues do not express in vitro (between Met-1 and Lys-75 exclusive). New upstream tail end primers were ordered to clone off the transformed pNIC vector with full insert. The gene sequence with the omitted leading codons will be copied off this template. (Both PCRs at 58° C and 61° C failed to produce bands on 1 kb ladder - image will come later) ==> Retry pNIC-Bsa4 cloning with perhaps undiluted KOD. Week 7 (blank gel) ==> Taking the most promising set of PCRs, PCR^2 was carried out and cleaned up. The nanodrop shows decent concentrations, but the get failed and had no bands, even the ladder was absent. The gel will be redone.
 * Rerun gel
 * gel check
 * tcDHFR project put to the side to join Kaarthik's and Andrew's pfDXR project!
 * [|New tail end primers for pfDXR]
 * Ran PCR off pNIC-Bsa4 with gene insert to clone truncated gene sequence.
 * gel check
 * PCR^2 clean up
 * nanodrop
 * gel check

Week 6 [1 kb ladder, 1°, 2°] ==> This gel again shows no real product of any use. A ghost band is seen on the first lane over from the ladder, but that product would be much too small. This set of PCRs does not seem viable, and PCR^2 should be carried on from the first set of PCRs from week 5 which yielded much better results.
 * (continue from PCRs)
 * gel check
 * PCR^2 (continue from first set of week 5 PCRs)

Week 5 [1 kb ladder, 1°, 2°] ==> The gel shows a distinct lack of bands. Gel should be rerun to recheck these PCRs, and new PCRs should be made simultaneously. [1 kb ladder, 1°, 2°] ==> This gel suggests that previous PCRs did yield product as opposed to the first gel without any bands. The 2° PCR has a single band at approximately the right size which looks promising, however, it is not a very strong band. New PCRs were made, so the next step would be to check those to see if they give better results.
 * Remade 20 µM tail end primer dilution (as per Dr. B's suggestion)
 * 2° PCR (continue from 1° PCR)
 * gel check 1
 * gel check 2
 * 1° and 2° PCR

Week 4
 * [|Virtual Screening Refresher]
 * New Oligo mix
 * 1° PCR

Week 3 [1 kb ladder, 1°, 2°, PCR^2] ==> Once again, the nanodrop results showed that there was a high concentration of DNA in the PCR^2 sample after clean up. The gel check still showed an unclean product with a large smear in the PCR^2 column as well as at least one contaminating band. The brighter band is about the size of the product. The next step is perhaps to redo the oligomix to see if better results can be obtained.
 * Retry PCRs (1°, 2°, PCR^2)
 * PCR^2 nanodrop
 * gel check

Week 2 == [1 kb ladder, 1°, 2°, PCR^2] ==> The nanodrop showed very good yields from clean up, however, after running the product on a gel, it is clear that the bands are way too short to be gene product. The weird banding pattern is also currently unexplained. The oligo mix is not expected to have a problem, so the PCRs should be redone from the mix as the next step.
 * PCR^2, clean up, and checks (continue from 2° PCR)
 * nanodrop
 * gel check

Week 1
 * [|PyMOL refresher]
 * 1° and 2° PCR (continue from summer oligomix)

=**Super Lack of Summer Stuff**= Sorry

070212 - Alex, your page is sad because it doesn't have any results to show! -- Dr. B

062112 - Alex - add your gel images and etc. in reverse chronological order. -- Dr. B

hello world!!

pGFP Nanodrop results