- which conjugal strains do you use in the lab? what plasmids do they have?
HB101, pRL443, pRL623(??)
- can we put in the required plasmids in other E coli strains if MC1061 is not available
- do the plasmids come with the strain or do we have to order them separately?
They have E coli HB101 containing pRL443 and pRL623
- have you used the cyanogate protocol before?
they are familiar with it but they advised us not to use it because it is a fairly complicated and time consuming procedure to clone just one gene
- Have you got a domesticated cscB?
- where do you buy the ingredients for BG-11 from?
HiMedia - 1 bottle should be sufficient to make 50L, we also need to add vitamin B12
- ashwin should show pics of the incubator and we can ask them how to fix LEDs
Fix lights to the panel above
Assemble gene, promo and terminator - amplify independently
overhangs for promoter, gene and terminator that overlap - join by Cpec or overlap extension PCR - makes casette
transform into dh5alpha and selection by PCR wrt to the transformed genes by using primers for em
PCR of plasmid alongwith restriction to confirm cassette in correct orientation
post meeting notes:
pSyn_1 and pSyn_6 are the integrative plasmids they have for PCC 7942, they both integrate into NS1, both have promoters and terminators, second has bom site, both have specR
we can amplify Pcpc560 from 6803 and a terminator from 7942 or 6803 and then assemble them together with cscB, we can create overhangs for all three parts so that they overlap and join - joining can be done by cpec or overlap extension PCR, we can synthesise these parts with overhangs if it is faster
the 5' end of promoter and 3' end of terminator can have restrictions sites so you can paste it into vector, you can have one more restriction site at 3' of promoter so that you can remove promoter and replace with other, then we can experiment with 3-4 different promoters to see how yield changes, selection of plasmids with successful cloning is done by PCR and restriction enzymes
It is best if our integrative suicide vector is small because larger ones are harder to transfer via conjugation
veeral will send photos of their set up
watch youtube videos on how to use SnapGene or Geneious
Come up with constructs by Sunday!!!!!