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Team:Lethbridge/Notebook/Planning
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<li>[[Team:Lethbridge/Notebook/Working_Glycerol_Stocks|Working Glycerol Stocks]]</li> | <li>[[Team:Lethbridge/Notebook/Working_Glycerol_Stocks|Working Glycerol Stocks]]</li> | ||
</ul> | </ul> | ||
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Work to be done: | Work to be done: | ||
| - | + | =Week of June 14/2010= | |
| - | + | ==Justin <i>et al</i>== | |
| - | + | ===Finish mms6-dT work=== | |
*Heat kill ligase | *Heat kill ligase | ||
*take small sample for gel | *take small sample for gel | ||
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*Run gel of above | *Run gel of above | ||
*Transform into DH5α | *Transform into DH5α | ||
| - | + | ===Test T4 DNA Ligase:=== | |
*<del>Restrict rbs-xylE with EcoRI and SpeI (take sample for analysis on gel)</del> | *<del>Restrict rbs-xylE with EcoRI and SpeI (take sample for analysis on gel)</del> | ||
*<del>Heat kill EcoRI and SpeI</del> | *<del>Heat kill EcoRI and SpeI</del> | ||
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*<del>Run unrestricted, restricted and ligated samples on a gel.</del> | *<del>Run unrestricted, restricted and ligated samples on a gel.</del> | ||
| - | + | ===Prepare plasmid DNA for sequencing=== | |
*<del>Adam to upload guidelines for preparation.</del> | *<del>Adam to upload guidelines for preparation.</del> | ||
| - | + | ===Test PCR conditions for confirmation of ligation via PCR:=== | |
*Run a PCR of lumazine synthase gene that was sent for sequencing (and subsequently confirmed) | *Run a PCR of lumazine synthase gene that was sent for sequencing (and subsequently confirmed) | ||
**using VF2 and VR primers | **using VF2 and VR primers | ||
*Run this PCR on a 2% agarose gel | *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. | 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== | |
*<del>Finish VWR order (1000µL tips); test tubes; test tube racks; 96 well plates for sequencing, petri dishes</del> | *<del>Finish VWR order (1000µL tips); test tubes; test tube racks; 96 well plates for sequencing, petri dishes</del> | ||
*<del>Get RMA for VWR order (ie wrong test tube and test tube racks)</del> | *<del>Get RMA for VWR order (ie wrong test tube and test tube racks)</del> | ||
Revision as of 03:53, 24 June 2010
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Back To:
Work to be done:
Contents |
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 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
