Team1Noteboook

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