Team:Newcastle/Labwork/8 October 2009

=Formal Lab Session - 8th October 2009=

Introduction

 * Today we did restriction digests again with pMUTIN4 with the new enzymes. We concluded that the enzymes we used may have been contaminated. We ordered normal new enzymes and used them instead.
 * We also did PCR fragment digests (from the cleaned PCR product) with BamHI and HindIII
 * We run the digestions on the gel and cut PCR fragment and pmutin4 from the gel to clean up.
 * Our B.subtilis transformation worked well yesterday, so we can perform new transformation using our pGFP-rrnB:kinA vector.
 * Also we set up mini and midi culture yesterday, we need to do mini prep and midi prep today.

pmutin4 digest
1.pmutin4 digest with HindIII and BamHI H2O 26ul 10X Buffer E 5ul BSA 0.5 ul (diluted) DNA 15ul HindIII 1ul BamHI 1ul

2.pmutin4 control digest with HindIII H2O 13.5 ul 10X Buffer E 1ul BSA 0.5 ul (diluted) DNA 3ul HindIII 0.5ul

3.pmutin4 control digest with BamHI H2O 13.5 ul 10X Buffer E 1ul BSA 0.5 ul (diluted) DNA 3ul BamHI 0.5ul

4.pSB1AT3 control digest with HindIII and BamHI H2O 13 ul 10X Buffer E 1ul BSA 0.5 ul (diluted) DNA 3ul HindIII 0.5ul BamHI 0.5ul

B.subtilis transformation

 * Follow the protocal from our lab protocol of B.subtilis transformation.
 * 5ul kinA DNA was used for transformation.

Midi prep cotC part

 * After the standared Midi procedure, we performed DNA concentration process and suspended midi DNA in 250ul PCR water.

Mini prep cotC and kinA culture

 * Followed Phil's Mini prep protocal.