Eric Wong's notebook

7/16 Dephosphorylation posted ‎‎Jul 23, 2009 11:11 AM‎‎ by ertwong@yahoo.com

We had to dephosphorylate the vector backbone so it doesnt close on itself, then pcr purify the sample.

7/15 Aar1 Digest posted ‎‎Jul 23, 2009 11:04 AM‎‎ by ertwong@yahoo.com

after w mini preped our transformations we digested each sample: Tuba Vav Vav Dock Itsn Sos1 Beta-pix As well as our backbone samples. Alw169 Oliver's sample

The digestion of the backbone samples turned out really clean, but on the other hand the digestion of our gpcr samples were a mess. Turns our some samples didnt cut (vav, beta-pix) Dock has Aar1 restriction sites inside the gene, sos1 and tuba has the same size as the topo vector( so it was hard to distinguish the difference between the two).

7/14 Blue/White screening posted ‎‎Jul 23, 2009 10:53 AM‎‎ by ertwong@yahoo.com

after a day of incubation we got the results of our topo cloned transformed cells. the one that did transform remained white, but the cells that did not transform turned blue. the only catch was the cells turn white even tho the vector enters the wrong way so you get a 50/50 chance of picking the right colonies for mini preps.

7/13 TOPO? posted ‎‎Jul 23, 2009 10:45 AM‎‎ by ertwong@yahoo.com

After jackie and katja pcr some of the GPCRs it so happens that the primer we ordered were wrong, so we had to resort to a method of cloning called TOPO cloning. in this method we use our sample and add a string of "A" nucleotides to each end allowing for ligation in a TOPO TA vector.

so our first step was to add the poly-A-tails, Runt he samples on a 50ml gel, perform gel extraction, and finally transformations with Blue and white screening.

7/6 New Cells posted ‎‎Jul 23, 2009 10:40 AM‎‎ by ertwong@yahoo.com

After working with tg1 cells for about a week and a half, we decided to work with dh5Alpha cells. the reason being was tg1 cells naturally has a large number to endonucleases and are hard to wash all of them off with pb and pe buffers. this opens the possibility of our DNA samples being cut, thus the switch to DH5 Alpha. Aynur says it has a better proficency rate as well has less endonucleases that tg1 cells.

7/1 First Screening posted ‎‎Jul 6, 2009 8:45 AM‎‎ by ertwong@yahoo.com

a majority of the first screening is rather disappointing since i added 500ul of eb buffer instead of 50ul and after nano dropping them the concentrations were rather low. we also started new transformation pow55 R1-25, pow646 02-19, Tiam-1, P-Rex 5

6/30 Transformations posted ‎‎Jul 6, 2009 8:36 AM‎‎ by ertwong@yahoo.com

After first round of transformations only 12 of our 21 transformations were successful. the reasons were 8 of the plasmids were plated on the incorrect plate resulting in no colonies a lawn, so we had to redo the ones that didnt work. meanwhile we start on our mini preps with 2 colonies per sample with their corresponding antibiotics..

6/29 GPCR Screening posted ‎‎Jul 6, 2009 8:25 AM‎‎ by ertwong@yahoo.com

Groups

GPCR's               Delta-opioid receptor parts                Actin-Related Parts GEF's                  Dor-villin                                            Act A 30-612 Beta-pix               Actinin                                               LPD (lamellipodia) Alpha-pix             Dor- Ezrin                                          EGFP-VASP Vav                      Dor-ERM                                           Act A 225-392 Tuba                    Dor- KIFC                                          GST LPD 755-1250 Dock 2                 Dor Tiam-1 Sos-1 Intersectin