Team:Brown/Notebook weekly Logs/Weekly Team1 Notebook

Week 3: June 29 – July 3, 2009

Monday Lab Meeting • Team 1 is working on digesting insert out of pBluescript using ECOR1 and BAMH1 • pVU2 Site→ use to analyze digest of plasmid • When cloning inserts into vectors: make sure insert sequence is in frame • Keep in mind the reading frame when cloning into pNO-TAT→ check for additional sequences on both ends of inserts

Adrian’s Restriction Digest Suggestions • 1 unit of enzyme cuts 1µg DNA ~1 hour • Use narrow wells with 0.5 µg DNA, wide wells with 4.5 µg DNA • Digest mastermix • Can cut with enzyme again if not sure whether plasmid

Week Summary

6/29 - Ran gel of last Friday’s troubleshooting digests performed on various plasmids to confirm enzyme efficiency -Recorded DNA concentrations for last Thursday’s minipreps→ medium-high concentrations -Re-ran digest since troubleshooting digest was successful→ unsuccessful digest -Plated out glycerol stocks→ Michael -Prepared minipreped pBluescript w/ insert samples for sequencing -Learnt how to properly use the nanodrop -PCR primers with restriction sites engineered arrived via FedEx

6/30 - PCR rxn of pBluescript w/ insert using primers that came in on 6/29 performed -Re-ran digest from 6/29 with great care; mastermix used -Sequences of pBluescript and pNOTAT examined to be in frame -Learnt how to re-suspend primers

7/1 - Ran improved PCR with adjusted temperatures and using Green Mastermix with Taq -New troubleshooting restriction digest performed: digested 5 different plasmids -Read binding affinity papers sent by Steph, and also read through pGEM vector kit protocol

Week 4: July 6 – July 10 2009

Monday Lab Meeting -Failed digests and PCR- could it be poor technique, or insert not in pBluescript? -Drop off fedex at Biomed front desk for sequencing; get package out to Yale right away -Hope to isolate EV131 insert at least by the end of the week, if not earlier

Suggestions: -Run lower percentage gel to get resolution between 3000 and 3500, usually run 1% gel -Run a couple of individual digests with individual enzymes -PCR: Amplify any other template alongside as a control

Week Summary

7/6 - Dropped of samples for sequencing - Ran digests of EV131 + individual enzymes (ECOR1, BAMH1, ECO&BAM, and “GUS” control plasmid + ECOR1) - Ran digest on a low resolution gel (1%-0.8%) to see difference between 3&3.5 kb (linearization of DNA) - Ran digests on other plasmids (“GUS” plasmid) to test enzymes - Ran PCR of pBluescript w/ EV131 insert with new primers (w/ restriction sites) - Ran control PCR of “GUS” plasmid alongside - Arian gave suggestions for improving PCR

7/7 - PCR run again; however, no EV131 expected product seen on gel -Liquid culture of cells containing pBluescript plasmid→ miniprep them tomorrow

7/8 - Notes on making sure insert if actually in the plasmid: either insert fail or primer fail - Will run these together:

   1) T7 w/ T3:   
             a) 50 bp→ no insert present
             b) 500 bp→ an insert is present, but wrong insert selected
             c) 800 bp→ correct insert selected
    2) M13 Forward w/ Reverse universal primers→ 570 kB
    3) GUS DNA w/ T3 & T7
    4) GUS DNA w/ specific primers

- Miniprep of overnight liquid cultures performed; DNA concentrations recorded