Team:Groningen/Labwork/10 September 2013

Mirjam

Genomic DNA extraction for ΔCheY and ΔDes.
PCR to obtain the ΔCheY and ΔDes from the genomic DNA extraction of the strains.

Claudio

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.

Sebas

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)