Revision as of 10:44, 15 September 2009

Meetings Calendar

Lab Work - 11/09/09

Metal Sensing Team

Introduction and Outline

So far we have transformed DH5-alpha E. coli cells with two synthesized BioBricks, which are the cotC-GFP-smtA and the kinA BioBricks. The cotC-GFP-smtA BioBrick is involved within the metal sensing sub-project whereas the kinA BioBrick is involved with the Stochastic Switch sub-project. The Metal Sensing team have since taken these transformant colonies and used them to set up LB+kanamycin cultures for mini-preps and midi-preps; these were then left overnight in the orbital shaking incubator and are awaiting processing today. However there are a range of tasks which need to be accomplished today; these are listed below:

Prepare Mini-preps of the kinA and cotC-GFP-smtA transformants
- Carry out the mini-prep processes
- Analyse the mini-prep DNA by treating with restriction enzymes.
PCR the cotC-GFP-smtA BioBrick with pMK15 and pMK16
- Set up the PCR
- Run the PCR for the correct duration
- Analyse PCR products through DNA gel electrophoresis
- Clean up PCR products if PCR reaction proves to be successful
Midi-prep cotC-GFP-smtA, kinA, pGFP-rrnB, pMUTIN4 and pSB1AT3
- Carry out the five midi-preps.
- Quantify the midi-prep samples
- Run the midi-prep samples on agarose gel through DNA gel electrophoresis
Run products from a PCR reaction involving arsR and cadA DNA
- Run PCR products on gel
- If successful, clean up fragments, run on gel again, cut with restriction enzymes NheI and BamHI, clean and then ligate together fragments.

Procedure

The following procedures were carried out:

Freeze the kinA and cotC-GFP-smtA transformant cells

Instead of preparing mini-prep samples from the two groups of transformant E.coli cells (i.e. kinA transformed E.coli cells and cotC-GFP-smtA transformed cells), we instead took the decision to use the inoculated 5ml LB tubes in freezing the cells. Analysis of the midi-prep samples will be used to determine whether the BioBricks have been truly transformed - this decision will both save time and also provide a back-up stock of cells for future work. This procedure was carried out by James using the protocol

PCR the cotC-GFP-smtA BioBrick with pMK15 and pMK16

Midi-prep cotC-GFP-smtA, kinA, pGFP-rrnB, pMUTIN4 and pSB1AT3

The midi-preps were conducted, as usual, using the Sigma-Aldrich Midi-prep kit and protocol. There were no changes to the protocol and 10ul samples of the five midi-prepped plasmids were loaded onto 0.8% agarose gel (see the 'Results' section below). The midi-prepped DNA samples were also assessed for DNA concentration using the NanoDrop spectrophotometer.

cotC-GFP-smtA

The following diagram shows the concentration of the cotC midi-prep sample; at 89.7 ng/ul the DNA concentration is at a suitable level for further processing.

Run products from a PCR reaction involving arsR and cadA DNA

Results

We took the decision to run 4 sets of DNA samples on 0.8% agarose gel - this includes the midi-prep samples (i.e. cotC, kinA, pGFP-rrnB, pMUTIN4 and pSB1AT3), products from a PCR reaction involving cotC, futher midi-preps of pMUTIN4 and digests from midi-prepped DNA the Stochastic Switch team have prepared (i.e. sac, ara and sleB) The resulting GelDoc photograph can be seen below:

Team Newcastle 2009 iGEM Geldoc 2009-09-11 15hr 59min.jpg

The lanes are as follows:
Row 1

Lane 1 = blank
Lane 2 = HindIII DNA Ladder
Lane 3 = cotC midi-prep sample
Lane 4 = kinA midi-prep sample
Lane 5 = pGFP-rrnB midi-prep sample
Lane 6 = pMUTIN4 midi-prep sample
Lane 7 = pSB1AT3 midi-prep sample
Lane 8 = blank
Lane 9 = cotC PCR reaction 1 sample
Lane 10 = cotC PCR reaction 2 sample
Lane 11 = cotC PCR reaction 3 sample
Lane 12 = cotC PCR reaction 4 sample
Lane 13 = blank
Lane 14 = pMUTIN4 midi-prep sample (from second attempt)
Lane 15 = leak-over of sample from lane 14
Lane 16 = pMUTIN4 midi-prep sample (from second attempt)
Lane 17 = HindIII DNA Ladder

Row 2

Lane 1 = blank
Lane 2 = HindIII DNA Ladder
Lane 3 = sac midi-prep digest sample
Lane 4 = ara midi-prep digest sample
Lane 5 = sleB midi-prep sample
Lane 6 = HindIII DNA ladder

Conclusions

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)

@@ Line 35: / Line 35: @@
 =====''cotC-GFP-smtA''=====
 [[Image:CotC Quantity DNA 11-09-09.JPG|550px]]
+The following diagram shows the concentration of the ''cotC'' midi-prep sample; at 89.7 ng/ul the DNA concentration is at a suitable level for further processing.
 ====Run products from a PCR reaction involving ''arsR'' and ''cadA'' DNA====

Team:Newcastle/Labwork/11 September 2009

From 2009.igem.org