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.

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

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