Team:Newcastle/Labwork/1 September 2009
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Contents |
Lab Session 01/09/2009
Promoter Library Sub-Project
Introduction
Up until now, this sub-project has seen the pGFP-rrnB plasmid backbone digested and linearised with restriction enzymes EcoRI and NheI. The resulting 8kb fragment (which is the plasmid backbone) has been successfully excised from agarose gel and purified; it is into this plasmid backbone in which we will place our sigA promoters.
Today's work involves the amplification of the promoter library (a collection of sigA promoters specified by degenerate sequences) using PCR.
Preparing the Primers
The first task carried out was the rehydration of the primers involved with this sub-project. A final concentration of 200uM is the desired value and to get this for each primer, the following calculation was observed:
Amount of water to be added (ul) = amount of primer (nMoles) x 5
The table below shows the oligos involved in this practical along with the primer amounts (in nMoles) and the amount of PCR water needed (in ul) to make the primers at a concentration of 200uM
Oligo | Purpose | Amount (nMoles) | PCR Water added (ul) |
---|---|---|---|
IDT 60396420 | Forward Primer | 19.6 | 98.0 |
IDT 60396419 | Reverse Primer | 21.9 | 109.5 |
alta D68423AW | Control | 0.06 | 300 |
alta D68425AW | Variant 2 (V2) | 0.06 | 300 |
alta D68424AW | Variant 3 (V3) | 0.06 | 300 |
alta D68426AW | Variant 1 (V1) | 0.06 | 300 |
alta D68427AW | 19mer Primer | 0.08 | 400 |
All of these oligos were then aliquoted and further diluted 1:100 with PCR water and stored in the -20C freezer. The primer stocks are kept in Neil's yellow box whereas the dilutions in enzymes are kept in the a separate box.
Preparing the PCR
The table below shows the amounts of substances added to each PCR reaction:
No. | Water (ul) | PCR Mix (ul) | Forward Primer (ul) | Reverse Primer (ul) | Template (ul) | Polymerase used (ul) | Total Volume (ul) |
---|---|---|---|---|---|---|---|
1 | 31 | 15 | 1 | 1 | 200uM (1ul) | Taq (1ul) | 50 |
2 | 31 | 15 | 1 | 1 | 200uM (1ul) | Taq (1ul) | 50 |
3 | 31 | 15 | 1 | 1 | 200uM (1ul) | Taq (1ul) | 50 |
4 | 31 | 15 | 1 | 1 | 200uM (1ul) | Taq (1ul) | 50 |
News
Events
- 20 – 21 June 2009 - Europe workshop (London)
- 23 – 24 June 2009 - UK iGEM meetup (Edinburgh)
- 23 October Practice Presentation (Newcastle)
- 23 October T-shirts are ready
- 27 October Practice Presentation (Sunderland)
- 27 October Poster is ready
- 30 October – 2 November 2009 - Jamboree (Boston)
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