Team:Newcastle/Labwork/7 October 2009

=Formal Lab Session - 7th October 2009=


 * We succeed in cotC transformation, lots of colonies grow on LB+Amp plates. We will culture the colonies for Mini prep and Midi Prep.
 * Even we got few colonies on LB+Amp plate for kinA transformation, we need to repeat transformation today and use LB + spectinomycin as antibiotic selection.



CotC PCR
We wanted to start from scracth for CotC biobrick. We will also PCR it. CotC 1ul of CotC biobrick synthesised in PMK 39ul of PCR water

GoTaq 2ul GoTaq 18ul H2O

PCR Mix: per tube: 10ul 5xGoTaq buffer prepared above + 5uk DNTP

Tubes(Prepared 6 tubes): 26ul H2O 15ul PCR mx 2.5 ul forward primer (It was diluted before) 2.5 ul reverse primer 2ul template prepared above (For the 6. tube, control tube, used water instead)

We setup 6 PCR reactions in two groups. Group 1 (3 tubes) 50C annealing temperature 90seconds extesnion time 31 cycles

Group 1 (2 tubes + control) 52C annealing temperature 90seconds extesnion time 31 cycles

we run the PCR products on the gel. run 8uk os PCR product from 6 tubes. The results were good for all tubes. We then cleaned the PCR products using the PCR clean-up kit.

Restriction Digest for pmutin4
We did restriction digest with two controls to see is the enzymes were working well. If they work we will clean the gel for pmutin4.

pmutin4 Digest with HindIII and BamHI H20 26ul 10x Buffer E 5ul DNA 15ul HindIII 2ul BamHI 2ul

pmutin4 Digest with HindIII only H20 15.5ul 10x Buffer E 2ul DNA 2ul HindIII 0.5ul

pmutin4 Digest with BamHI only H20 15.5ul 10x Buffer E 2ul DNA 2ul BamHIIII 0.5ul

Mixed the tubes well and incubated for three hours at 37C

In the meantime prepared 1xTAE buffer and 0.8% agarose gel.

Run the digests on the gel Digest1:50ul from the digest + 10ul 10X loading buffer Digest2:9ul from the digest + 1ul 10X loading buffer Digest3:9ul from the digest + 1ul 10X loading buffer

pmutin 4 digest results were not very clear!

E.coli Transformation

 * Since we used wrong medium for yesterdays E.coli transformation, there was no colony grow for kinA transformation, so we redo E.coli transformation today.
 * 1ul pGFP-rrnB:kinA vector was used for E.coli transformation.
 * All transformation cells were sprayed on LB+Spec plates

B.subtilis transformation

 * To save our kinA DNA and to make sure that our B.subtilis transformation work, we did a B.subtilis transformation with pure pGFP-rrnB plasmid.

1. 0.4ml B.subtilis cell  +  pGFP-rrnB      5ul 2. 0.4ml B.subtilis cell  +  pGFP-rrnB      5ul 3. 0.4ml no cell control  +  water          5ul 4. 0.4ml B.subtilis cell  +  water          5ul


 * All transformed cells were sprayed on LB+CHL plates (CHL concentration 5ug/ml)
 * Yesterday's transformation cells left were sprayed on LB+CHL plates (CHL concentration 5ug/ml)

Culture cells for mini and midi prep

 * Picked up colonies for cotC transformation.
 * Picked up colonies growed on LB+Amp for kinA transformation.
 * 5ml LB+Amp/ LB+Spec liquid medium were used for culture.
 * 50ml liquid LB+Amp medium for cotC midi prep culture.