Team:Newcastle/Labwork/1 September 2009
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=Lab Session 01/09/2009= | =Lab Session 01/09/2009= | ||
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==<u>Stochastic Switch Team</u>== | ==<u>Stochastic Switch Team</u>== | ||
[[Image:Team Newcastle 2009 iGEM IMG 0586.JPG|200px|thumb|Goksel cleaning up products from a successful PCR reaction involving ''sspB'']] | [[Image:Team Newcastle 2009 iGEM IMG 0586.JPG|200px|thumb|Goksel cleaning up products from a successful PCR reaction involving ''sspB'']] |
Revision as of 00:13, 17 September 2009
Lab Session 01/09/2009
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
Today we also made ON cultures of the following strains in order to freeze them down into the TPA collection. First the TPA book had to be filled out to record the strains:
- 2489- BBa_CO178
- 2490- BBa_ROO62
- 2491- BBa_CO161
- 2492- BBa_CO179
- 2493- BBa_ROO79
- 2494- BBa_J44000
These will be frozen down in DMSO tomorrow.
Metal Sensing Team
Today we will carry out PCR in order to amplify czrA gene for our team and sac, ara and sspb genes for the stochastic switch team.
PCR
We carried out PCR reactions in the same way as described on 21/08/2009 along with the stochastic switch team.
No. | Water (ul) | PCR Mix (ul) | Forward Primer (ul) | Reverse Primer (ul) | Template (ul) | Polymerase used (ul) | Total Volume (ul) |
---|---|---|---|---|---|---|---|
1 | 24 | 15 | pMA1 2.5ul | pMA2 2.5ul | 168 DNA 5ul | MolTaq (1ul) | 50 |
2 | 29 | 15 | pMA1 2.5ul | pMA2 2.5ul | 0 | MolTaq (1ul) | 50 |
3 | 24 | 15 | sac_forward 2.5ul | sac_reverse 2.5ul | 168 DNA 5ul | MolTaq (1ul) | 50 |
4 | 29 | 15 | sac_forward 2.5ul | sac_reverse 2.5ul | 0 | MolTaq (1ul) | 50 |
5 | 24 | 15 | araR_forward 2.5ul | araR_reverse 2.5ul | 168 DNA 5ul | MolTaq (1ul) | 50 |
6 | 29 | 15 | araR_forward 2.5ul | araR_reverse 2.5ul | 0 | MolTaq (1ul) | 50 |
7 | 24 | 15 | sspb_forward 2.5ul | sspb_reverse 2.5ul | 168 DNA 5ul | MolTaq (1ul) | 50 |
8 | 29 | 15 | sspb_forward2.5 | sspb_reverse 2.5 | 0 | MolTaq (1ul) | 50 |
PCR conditions:
For sample 1 and 2:
1- 95C for 2min 2- 95C for 30sec 3- 63C for 1min 4- 75C for 2min Step 2-3-4 x 30 5- 75C for 5min 6- 4C Pause
For sample 3, 4, 7 and 8:
1- 95C for 2min 2- 95C for 30sec 3- 57C for 1min 4- 75C for 2min Step 2-3-4 x 30 5- 75C for 5min 6- 4C Pause
For sample 5 and 6:
1- 95C for 2min 2- 95C for 30sec 3- 59C for 1min 4- 75C for 2min Step 2-3-4 x 30 5- 75C for 5min 6- 4C Pause
Then run the PCR prodcuts on a gel. Results:
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 (C0) | 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:
Sample | No. | Water (ul) | PCR Mix (ul) | Forward Primer (ul) | Reverse Primer (ul) | Template (ul) | Polymerase used (ul) | Total Volume (ul) |
---|---|---|---|---|---|---|---|---|
C0 | 1 | 31 | 15 | 1 | 1 | 200uM (1ul) | Taq (1ul) | 50 |
V1 | 2 | 31 | 15 | 1 | 1 | 200uM (1ul) | Taq (1ul) | 50 |
V2 | 3 | 31 | 15 | 1 | 1 | 200uM (1ul) | Taq (1ul) | 50 |
V3 | 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.
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]