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Team:Lethbridge/Notebook/Planning
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Adam.smith4 (Talk | contribs) (→Week of June 14/2010) |
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*Ligate with T4 DNA Ligase | *Ligate with T4 DNA Ligase | ||
*Run unrestricted, restricted and ligated samples on a gel. | *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:===== | =====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) | ||
| Line 71: | Line 73: | ||
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==== | ====Adam==== | ||
| - | *Finish VWR order (1000µL tips); test | + | *Finish VWR order (1000µL tips); test tubes; test tube racks; 96 well plates for sequencing |
*Get RMA for VWR order (ie wrong test tube and test tube racks) | *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. | ||
Revision as of 02:52, 12 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 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
- 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.
