Team:Newcastle/Labwork/22 September 2009

=Formal Lab Session - 22nd September 2009= = Overview = 
 * Metal Sensor Team - 
 * Stochastic Switch Team - 
 * Sporulation Tuning/Chassis Team - 

Metal Sensor Team
We carried out the following tasks: '''Please note there were three reactions: 1) vector + insert + ligase, 2) vector + ligase and 3) vector + NO ligase.
 * Digested cotC-GFP-smtA PCR products with BamHI and HindIII (FastDigest) and allowed them to incubate for 1 hour under 37C.
 * Conducted PCR clean-up using GenElutes PCR clean-up kit
 * Digested cotC-GFP-smtA with BamHI and HindIII
 * Carried out FastLigations with the following concentrations:
 * 1) 2ul of ligase buffer
 * 2) 2.5ul of vector DNA (i.e. pMUTIN4)
 * 3) 14.5ul of insert DNA (i.e. cotC-GFP-smtA)
 * 4) 1ul of ligase
 * We also started to make some competent E. coli cells by carrying out the 'Culture' step in Phil Aldridge's preparing competent cells protocol

Stochastic Switch Team
Today we transformed E.coli JM109 cels with the overnight ligation product from yesterday using the promega protocol. We used the following controls:


 * Tube 1: Backbone + insert + ligase (proper ligation)
 * Tube 2: Backbone + ligase (control for background ligation level)
 * Tube 3: Backbone + insert (control for ligase effectiveness)

These were left in the incubator for an hour and then plated out (2 plates per tube 200ul and 50ul) onto Amp + Tet LB plated and put in the 37C incubator overnight.

Today again we redid the digests of the sac miniprep however again there was no DNA present.

Introduction

 * After overnight culture of cells for Midi Prep, today we need Midi Prep L1 and L2 plasmid and digest the plasmid to check the Midi prep result.

Midi prep L1(pSB1AT3:cwlJ) and L2(pSB1AT3:cwlJ:sleB)

 * Before we carried out Midi Prep, we take out 1ml of cell mix and put them into 1.5ml eppendorf tube to keep in fridge.
 * 50ml of cells were used for Midi Prep.
 * Midi prep processed by following the standard protocal of Plasmid Midi Prep Kit(Sigma).

Digest Midi prep result

 * Using the same reaction volume as day 18/09/09

Conclusion
cwlJ:sleB fragment 53.5ng/ul (after PCR using primer cwlJsleBForward and cwlJsleBReverse) pMutin4 after digestion with HindIII and BamHI 24.9ng/ul
 * Both Midi prep for L1 and L1 are right plasmid.
 * Nano dorp of our fragments and plasmid.

L1 Midi (plasmid pSB1AT3:cwlJ) 76.9ng/ul

L2 Midi (plasmid pSB1AT3:cwlJ:sleB) 55.0ng/ul

kinA fragment after digest(EcoRI and NheI) and gel extraction 6.1ng/ul