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Team:Newcastle/Labwork/1 October 2009
From 2009.igem.org
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Formal Lab Session - 1st October 2009
Restriction digest tests
Our previous transformations did not work well. We tested if we had the right pmutin4 and cotC biobricks.
We run them on the gel.
pmutin4:
4ul DNA 5ul Water 1ul loading buffer
CotC:
3ul of DNA 6ul DNA 1ul loading buffer
The bands were right. However some BamHI and HindIII sites on pMUTIN4 are too close, the backbone may not have been cut properly.
Preparation of competent cells
We prepared fresh DH5 alpha cells by plating them into two new LB plates
We also prepared a set of new E.coli DH5 alpha competent cells. we measured the ODs of E. coli cells during transformation. We waited 2 hours before measrung the ODs and we get the absorbance result as 0.146. To measure the ODs
Set the device to 600nM Place a tube with LB only and take a reference measurement Place the culture and press the green box.
We then tested our competent cells for transformation. We used 40ul of cells + 3ul DNA for the tests. We used pmutin4 and pSB1AT3 for the tests
Ligations
We prepared ligations for Cot + pmutin4. Used two different versions of pMUTIN4 and prepared the ligations below
- cotC + pMUTIN4 cut1
- cotC + pPMUTIN4 cut2
- psc + pMUTIN4 cut 2
We also prepared controls as below
- backbone + ligase + no insert
- backbone + no liase + no insert
Chassis team
Introduction
- Digest pMK-RQ:kinA and pGFP-rrnB:kinA with same enzyme and run DNA on agarose to check whether our Midi prep is the right one.
Experiment procedure
Digest pMK-RQ:kinA and pGFP-rrnB:kinA
- EcoRI and NheI were used for digestion.
dd H2O 7ul
10X fast digest buffer 2ul
Fast EcoRI 0.5ul
Fast NheI 0.5ul
pMK-RQ:kinA 10ul
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20ul
dd H2O 7ul
10X fast digest buffer 2ul
Fast EcoRI 0.5ul
Fast NheI 0.5ul
pGFP-rrnB:kinA 10ul
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20ul
- 37C 1 hour.
- Run the sample on 0.8% agarose gel.
Conclusion
- The gel picture showed that our kinA clone was not right.
lane 1: lamda ladder lane 2: 1kb ladder lane 3: pMK-RQ:kinA lane 4: pGFP-rrnB:kinA (our NO.6 in transformation)
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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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