Team:Newcastle/Labwork/1 September 2009
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Today we run the gel for pSB1AT3 plasmid backbone purified last week from the gel. It was cut with EcoRI and SpeI sites. The length of the band confirmed that we have the right product. | Today we run the gel for pSB1AT3 plasmid backbone purified last week from the gel. It was cut with EcoRI and SpeI sites. The length of the band confirmed that we have the right product. | ||
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We also used PCR clean-up kit to extract sspB from last week's PCR. We used sigma-d's PCR clean-up protocol. The final volume was measured as 48ul. We measured the concentration and run it on the gel using 5ul of sspb + 4ul of water + 1ul of the loading buffer. The band confirmed that we had the right product after the clean-up process. | We also used PCR clean-up kit to extract sspB from last week's PCR. We used sigma-d's PCR clean-up protocol. The final volume was measured as 48ul. We measured the concentration and run it on the gel using 5ul of sspb + 4ul of water + 1ul of the loading buffer. The band confirmed that we had the right product after the clean-up process. |
Revision as of 12:30, 2 September 2009
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 |
The PCR Mix was composed of the following substances:
- 30ul dNTP's (6 x 5ul)
- 30ul Buffer (6 x 5ul)
- 30ul DMSO (6 x 5ul)
The Taq polymerase used needed to be prepared too. 6ul of pure Taq enzyme was taken and 1ul was diluted in 5ul of PCR water. 1ul of this diluted Taq was used per tube.
Carrying out the PCR
The cycle for the PCR was as follows:
One cycle involving the reverse primer pGFGrrnBrev only with the large degenerate oligo as a template - this is to convert the single-stranded oligo into double stranded DNA. To denature the oligo 90C was needed, to anneal the oligo 60C was needed and to extend the oligo 72C was needed.
The forward primer should then be added and the mixture given 30 cycles (each cycle lasting 1 minute) of 90C denaturing, 54C annealing and 72C extending.
Stochastic Switch Team
Today we run the gel for pSB1AT3 plasmid backbone purified last week from the gel. It was cut with EcoRI and SpeI sites. The length of the band confirmed that we have the right product.
We also used PCR clean-up kit to extract sspB from last week's PCR. We used sigma-d's PCR clean-up protocol. The final volume was measured as 48ul. We measured the concentration and run it on the gel using 5ul of sspb + 4ul of water + 1ul of the loading buffer. The band confirmed that we had the right product after the clean-up process.
The concentration of sspB was 37.5ng/ul
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)
Social Net
- Newcastle iGEM Twitter
- [http://www.facebook.com/home.php#/group.php?gid=131709337641 Newcastle on Facebook]
- [http://www.youtube.com/user/newcastle2009igem Newcastle Youtube Channel]