Remember timings!
Stocks/kits/plates
require access to ULT freezer (let me know in advance)
material to autoclave needs to be ready before 12
Plasmids:
pUC19 needed for cloning AND as negative control in all transformation steps
pSDC115 needed as positive control
E.coli strains:
DS941 regular cloning (LB)
DS945 dimerisation (LB)
FC33/pSDC115 contains plasmid with two cer-site (to be used in week 3)
DS956 and DS957 (to be used in week 3 or 4)
Monday 23rd
Morning:
BEFORE 12: prepare things to be autoclaved (Arbaaz, Karman and Aramide, you will need LB media and LB/Amp plates)
Arbaaz, Karman and Aramide: start restriction digestion of pUC19 (you need to digest 10ul – ask the MSc students to give you an aliquot of their “Miraprep” sample, which you will need to dilute 100 times before using it) and of your cer-site (5ul – you need to resuspend this in sterile water first, please ask one of your friends to show you) with EcoRI and HindIII. Once you have started the digestion of the pUC19 plasmid, prepare an agarose gel according to the protocol you have and let it set – please add GelRed to the agarose in this case, we will make the elution under UV light to make sure you have enough DNA. When the digestion is done, deactivate the enzymes at 65C as written in the protocol, and load the samples on the gel – you can take a 45 break at this point.
For those of you who do not have colonies on LB/Amp for the cells transformed with the ligation: repeat the ligation reaction as you did on Thursday, but this time leave the reaction to incubate longer on the bench (possibly over night)
I will only be able to pass by the lab briefly around 11:00 in between two teaching sessions
Afternoon (starting at 13:30):
For those of you who have colonies on LB/Amp for the cells transformed with the ligation: start a liquid inoculum of transformed cells in small bottles of LB/Amp (two colonies from the transformation done with the ligation, and one colony from the positive control transformed with pUC19, if available – remember to add the antibiotic in each LB bottle!)
For those of you who do not have colonies on LB/Amp for the cells transformed with the ligation: you can try to repeat transformation with competent cells prepared by the technicians, if these are available (you’ll need to wait for me to ask for the aliquots, please do not do it yourself) OR with some of the aliquots you have left from last week
Arbaaz, Karman and Aramide: you will need to elute the pUC19 digested with the two enzymes from the gel according to the protocol of the kit. After this, you’ll need to set up a ligation and leave it on the bench overnight.
Start liquid inoculum of DS941 and DS945 to make competent cells (one 20ml LB bottle each)