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Team:Lethbridge/Notebook/Planning
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Work to be done:
Contents |
Week of June 14/2010
Justin et al
Finish mms6-dT work
Heat kill ligasetake small sample for geltake another sample for restriction (cut out entire biobrick)Run gel of aboveTransform into DH5α
Test T4 DNA Ligase:
Restrict rbs-xylE with EcoRI and SpeI (take sample for analysis on gel)Heat kill EcoRI and SpeILigate with T4 DNA LigaseRun unrestricted, restricted and ligated samples on a gel.
Prepare plasmid DNA for sequencing
Adam to upload guidelines for preparation.
Test PCR conditions for confirmation of ligation via PCR:
- Run a PCR of lumazine synthase gene that was sent for sequencing (and subsequently confirmed)
- using VF2 and VR primers
- Run this PCR on a 2% agarose gel
PCR Confirm previous ligations
If (AND ONLY IF) PCR conditions amplified our known lumazine synthase gene, set up PCR of our previous ligation reactions to see if there is any ligated product.
Adam
Finish VWR order (1000µL tips); test tubes; test tube racks; 96 well plates for sequencing, petri dishesGet RMA for VWR order (ie wrong test tube and test tube racks)- Get guidelines for sequencing.
Make primers for inserting mms6, xylE, lumazine, (C and N terminal labelled CFP/YFP-maybe) into pET28a expression vectors.No longer required.Order polymerase, SpeI, NotI, and (maybe, depending on results) T4 DNA ligase from Fermentas.Check with HJ's lab to see which polymerase they use and from whom.
- Restrict lumazine, mms6, and xylE from BioBrick plasmid with NotI, and ligate into pET28a plasmid (pre-cut with NotI).
- Check ligation and gel extract bands of proper size (Check for double insertion, plasmid self-ligation)
- Transform into DH5α and purify plasmid DNA
- Perform restriction analysis (using EcoRI and EcoRV) to check orientation of insert
- Perform overexpression tests
- Send for sequencing
Week of July 5th/2010
One
1) Maxiprep cells with the following BioBricks:
*pLacI
*pBad
*dT
*CFP compete
*Mms6 Mr. Gene
*pBad-TetR
*Lumazine
*EYFP
2) Request parts from the Registry
3) Send in for sequencing the products of PCR from June X/2010
4) Come up with PET28a plan B
5) Put flow chart on wall
Two
1) Add dT (from maxiprep above) to
Mms6- xylE
Lumazine synthase
All above already maxiprepped
2)Overexpression test of "CFP Complete" and mms6
Three
- Restrict lumazine, mms6, and xylE from BioBrick plasmid with NotI, and ligate into pET28a plasmid (pre-cut with NotI).
- Check ligation and gel extract bands of proper size (Check for double insertion, plasmid self-ligation)
- Transform into DH5α and purify plasmid DNA
- Perform restriction analysis (using EcoRI and EcoRV) to check orientation of insert
- Perform overexpression tests
- Send for sequencing
Week of July 12th/2010
One
1) Add dt to Mms6,Lumazine, and xylE
- Restrict Individual Parts
- Set up ligation reaction
- Confirm ligation using PCR
- Transform confirmed parts into DH5α
- Miniprep confirmed parts
2) Maxipreps
- pLacI
3) Jeff to finalize signal sequences by Wednesday for Friday order
4) Friday order for four fluorescent proteins
- CEYFP
- NEYFP
- CECFP
- NECFP
5) Successful meeting with Bob
6) Adam to talk to Lisza about Mms6
7) Adam to compile list of confirmed parts for Lisza
8) Transform Mms6 into BL21(DE3)
9) Justin/Mackenzie to talk to David about working with Argon to make nanoparticles
10) Send away all maxipreps for sequencing
11) Quantify maxipreps using gel method
12) Talk to Hayes lab about borrowing some Argon that they have on tap..
Two
1) maxiprep
- pET28(a)
2) Assemble
- pLacI-sRBS
Three
1) Assemble
- pLacI-sRBS-lumazine-dt
- pLacI-sRBS-xylE-dt
- pLacI-sRBS-Mms6-dt
2) Insert Mms6, xylE, and Lumazine Synthase into pET28(a) over-expression vector
