Team:Newcastle/Labwork/23 September 2009

=Formal Lab Session - 23rd September 2009= = Overview = 
 * Stochastic Switch Team - 
 * Sporulation Tuning/Chassis Team - 

Stochastic Switch Team
Today we discovered that the transfrormations seemed sucessful, and the control plates had no growth at all which is what was hoped for. Hopefuly this means that we have a transformed ara-sspb-pSB1AT3 ligation product.


 * We set up miniprep cultures for 12 colonies using LB + amp + tet
 * We also set up 12 more miniprep cultures for the Sac transformed cells in hope that the precious minipreps had been carried out incorrectly.

Introduction

 * Today's main work is to ligate kinA with pGFP-rrnB vector and transform ligated vector into E.coli.

ligate kinA and pGFP-rrnB
dd H2O                                14.5ul Fast ligation buffer                     4ul pGFP-rrnB backbone(180ng/ul)           0.5ul kinA fragment(6.1ng/ul)                 15ul T4 ligase                                1ul -                                          35ul
 * ligate reaction


 * Votex for few seconds.
 * 22C 10min and 1 hour in RT.

Digest pMK-RQ:kinA and pGFP-rrnB
dd H2O                    7ul 10X fast digest buffer    7ul Fast EcoRI                3ul Fast NheI                 3ul pMK-RQ vector            50ul --                           70ul dd H2O                    7ul 10X fast digest buffer    7ul Fast EcoRI                3ul Fast NheI                 3ul pGFP-rrnB vector         50ul --                           70ul
 * pMK-RQ digest reaction.
 * pGFP-rrnB digest reaction.


 * Incubate 1 hour at 37 degree.

Purify the digest fragment and backbone

 * Run the digested DNA on big well agarose gel.
 * cut the right bands.
 * Use gel extraction kit to purify the DNA fragments.

Transformation

 * Followed the Phil's transformation protocal.

Make LB+Spectinomycin plates

 * The spectinomycin solution in frige is 20mg/ml
 * The concentration needed in plate is 50ug/ml
 * Add 2.5ml spectinomycin solution into 1 liter LB agar medium and pour the plate



Further plan

 * If th transformation was success, we need to prepare the Mini and Midi culture.
 * Collect the plasmid with kinA and do transformation for B.subtilis.