"

From 2010.igem.org

[http://2010.igem.org/wiki/index.php?title=Team:Lethbridge&Home ] [http://2010.igem.org/Team:Lethbridge/Team ] [http://2010.igem.org/Team:Lethbridge/Project ] [http://2010.igem.org/Team:Lethbridge/Notebook ] [http://2010.igem.org/Team:Lethbridge/Parts ] [http://2010.igem.org/Team:Lethbridge/Modeling ] [http://2010.igem.org/Team:Lethbridge/Ethics ] [http://2010.igem.org/Team:Lethbridge/Safety ]

Back To:

• Lab Work
• Calendar
• Protocols
• Working Plasmids
• Working Glycerol Stocks

Work to be done:

Contents 1 Week of June 14/2010 1.1 Justin et al 1.1.1 Finish mms6-dT work 1.1.2 Test T4 DNA Ligase: 1.1.3 Prepare plasmid DNA for sequencing 1.1.4 Test PCR conditions for confirmation of ligation via PCR: 1.1.5 PCR Confirm previous ligations 1.2 Adam 2 Week of July 5th/2010 2.1 One 2.2 Two 2.3 Three

Week of June 14/2010

Justin et al

Finish mms6-dT work

• Heat kill ligase
• take small sample for gel
• take another sample for restriction (cut out entire biobrick)
• Run gel of above
• Transform into DH5α

Test T4 DNA Ligase:

• Restrict rbs-xylE with EcoRI and SpeI (take sample for analysis on gel)
• Heat kill EcoRI and SpeI
• Ligate with T4 DNA Ligase
• Run 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 dishes
• Get 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