Team:Groningen/Labwork/10 September 2013



Genomic DNA extraction for ΔCheY and ΔDes.
PCR to obtain the ΔCheY and ΔDes from the genomic DNA extraction of the strains. PCR results showed that there are bands at around 500-750 bp, which we did not expect.
A new PCR reaction is made, using the genomic DNA of wild type B. subtilis 168 as control.


pSB1C3-S1-S5-S13 and pSB1C3-S2-S5-S14 plates showed colonies.
ColonyPCR on 5 colonies per plate was performed using VF2 and VR primers (annealing temperature 58°C).
The samples were checked on agarose gel 0.8% beside the positive control (pSB1C3-S1-S5), which is not shown in the picture.

All the colonies screened were positive candidates. The bands were all around the expected height: 1075 bps ('I am gonna get lucky' cit.).
Colonies C and D from pSB1C3-S1-S5-S13 were inoculated in LB + Cm and incubated over night (Sander).
Colonies A and D from pSB1C3-S2-S5-S14 were inoculated in LB + Cm and incubated over night (Sander).

The ligation products pSB1C3-S16-S3 and pSB1C3-S16-S9 were transformed into E. coli DH5α, plated on LB + Cm and incubated at 37°C over night.


All plates (containing 5µg/ml cm) show a smear of 'death' cells. One single colonies were only visible on the non-control plates. For GFP0840 there were ~20 colonies for RFP 1 colonie and for DSMgfp no colonie. Restreaked colonies on LB/Starch/CM plates.

Did digestion check on GFP0840 1&3, GFPdsm 1&3, RFP 1&4 with XhoI (digest in promoter and outside)
When promoter would not be there XhoI would cut 1 time (~8,5k), when promoter is there it would cut twice (~5k & 4k).

GFP0840 1  &  3      GFPdsm 1  &  3              RFP 1  &   4              1*              M
M: 10k, 8k, 6k, 5k, 4k, 3,5k, 3k, 2,5k, 2k, 1k, 750, 500.
1*: undigested RFP1
Promoter is in every construct except for RFP 1 (Did not digest at all)

Streaked 3 colonies of GFP840 and 1 colonie RFP on LB/starch/cm and LB/cm plates.