Formal Lab Session - 21st September 2009
Stochastic switch team
Today we digested the sac minipreps with EcoRI and XbaI. It seems clear that there was something wrong with the minipreps (such as poor procedure) as there seemed to be little or no DNA in the sample.
Did a gel fragment clean up on the sspb fragment cut from the gel, this was put in the 'fragments' box in the freezer ready for later.
50ul of ara+pSB1AT3 plasmid was cut with SpeI and PstI in order to insert the sspb fragment after the operator. Rather than doing a gel fragment prep a PCR clean up (kit) was done on the digest to save time.
We ligated the ara + sspb fragments using Chris' method:
- 2ul Ligase buffer
- 2.5ul Vector DNA
- 14.5ul insert DNA
- 1ul ligase
= 20ul
This was left in the fridge overnight to improve the ligation conditions.
Metal Sensing Team
Introduction
Practical Outline
Here are the list of tasks which need to be accomplished by the end of the day:
- Run analysis digests of the 5 midi-preps of cotC, kinA, pGFP-rrnB, pMUTIN4 and pSB1AT3 (midi-prepped in the 11/09/09 lab session and digested in the 18/09/09 lab session)
- If successful, double digest 40ul of pMUTIN4 with BamHI and HindIII and place under DNA gel electrophoresis; excise band produced and extract DNA
- If unsuccessful, inoculate 5ml of LB with E. coli containing pMUTIN4 (for mini-preps and midi-preps)
- Check PCR reaction (amplification of cotC-GFP-smtA) on gel and clean up if successful
- If successful, digest with BamHI and HindIII
- Should the pMUTIN4 DNA extraction be a success and the cotC-GFP-smtA BioBrick be successfully amplified (by PCR) and extracted, ligate the two fragments together.
- Reactions include:
- vector + insert + ligase
- vector + ligase
- vector and NO ligase
- Inoculate 3ml of LB with TPA2 cells (for production of competent DH5-alpha E. coli cells)
Procedure
Analysing midi-prep digests
Double digesting pMUTIN4 midi-prep DNA
Cleaning up cotC PCR product
Excising digested pMUTIN4 from gel
Quantification of cotC and pMUTIN4
Chassis team
Introduction
Experiment procedure
1
2
3
Conclusion
[[Image:|400px|center]]
Futher plan
July
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