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Team:Brown/Notebook weekly Logs/Weekly Team1 Notebook
From 2009.igem.org
Histamine Binding Protein Weekly Lab Log
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
7/9
- PCR using T7/T3 & M13(F) & R universal primers on GUS control
- 169 bp is expected size of the band will be if there is no insert, 741 bp is expected size if insert is present
- Expected band observed!
