*For posts on Salaita lunch and pictures of the end of organic chem 222 class of 2013, see below.
((These posts I'm writing for my own accounting on advice from a post-doc I knew who told me it was a good idea. However, I will also be putting them up here, for anyone who wants to know what I've been up to.))
Started working last Wednesday – it is now Friday – three days.
Watched Kevin perform one of his nanowalker experiments. Got tips and he showed me the pH meter and some things around the lab. Kevin showed me how to buy things from the stock room and picked me up a lab book.
Was able to get permission from I-Lin, Chris and Dr. Conticello to do electroporation transformations and protein expression in their lab. Read and wrote protocols for the week. Spent about an hour looking around the lab, marking where stuff was. Asked Kevin questions and got enough information for transformation and protein expression. Purchased gloves ($10.87).
Made an ampicillin 100 mg/ml stock. Purchased a 250 ml and 500 ml wash bottle and one blue rack (total $21.45). Did transformation of pQE-70/rtcB::Amp into Keio ΔrtcA::kan electrocompentent cells in the Conticello lab. I-Lin watched me do it and gave me helpful tips (see p. 5). Talked to Ashley in the Lutz lab about protein expression and got a few more specifics about the protocol (see p. 6). She gave me some Amp/Kan plates to plate the transformation on, so I could avoid re-streaking to a kan plate. I-Lin says that she’s free on Tu at 3 p.m. to show me the protein purification w/ the Ni-affinity column.
Purchases for the week = $32.32 + lab notebook cost
Yoshi said it was a good idea to buy a rack if I didn't have one, but I totally forgot about the racks in Kevin's drawer in the cold room - next time - I'll make sure to look around harder.
What I Still Need to Do
1. Take out the transformation plates in the 37C in Lutz lab tomorrow. Store in 4C in Salaita lab.
2. Read Mini-PROTEAN Tetra Cell booklet about SDS-PAGE gels today.
3. Read the QIAexpressionist handbook over the weekend.
4. Double check ligation experimental design and what the reaction mixture ought to be. Re-read Raines supplemental for that, as well.
5. Read the gel papers, to determine if it’s possible to make a gel that will separate 10mer and 20mer products.
6. Ask I-Lin about using the Bradford assay while I’m sonicating
7. Monday, prepare the reagents for the protein purification. Make a kanamycin stock. Find out whose centrifuge I can use, how to use the centrifuge, etc. Preferably find one that centrifuges falcon tubes. I feel safer about those being sterile.
8. Find out how to add in GBP to the plasmid. Read the GBP papers that I have concerning that and look up the ones about binding affinity that I found references for.
Tentative Schedule for Next Week
Friday – Prepare Amp and Kan stocks; transformation
Monday – Inoculate culture; prepare reagents
Tuesday – protein expression; purification via Ni-column; spin-column; 4C/-80C
Wednesday – SDS-PAGE gel to determine purity
Thursday/Friday – attempt ligation reaction