Transforming bacteria

Competent cells:

1) Innoculated a 5ml LB culture in 15ml tube with a DH5α colony. Grown overnight in 37°C incubator. Not shaking (might be better if it was). 2) Transferred the 5ml O/N culture into an 'autoclaved' conical flask containing 100ml LB broth and back into the 37°C incubator for about an hour. Then onto hot plate/,magnetic stirrer with a water bath on top to buffer the temperature a bit, it seemed easiest to keep the temperature stable at around 34 to 36°C, not ideal but it worked this time. 3) Cells allowed to grow until cloudy (much cloudier than last time), it would be nice to be able to quantify this! The closest I can get is to say that you can only see the magnetic stirring flea when it's on the near side of the flask, so almost opaque. This took about 3 hours.

note: Through steps 1 to 3 maintained aseptic conditions by doing all transfers etc under the gas flame.

4) Flask put into ~0°C water in polystyrene box with cold packs from the freezer plus ice added to the water, held there for 30 mins.

note: Cells after this point kept as close to the 0 to 4°C temp range as possible throughout the process by: i) 15ml/1400rpm centrifuge stored in fridge overnight. ii) rotor of microfuge kept in fridge overnight plus returned to fridge between steps. iii) All plastic consumables used were equilibrated to ice cold temp in the ice/ice pack bath at least an hour beforehand. iv) All reagents (MgCl2, CaCl2 and CaCl2 with 15% Glycerol) kept in fridge overnight and then moved to ice bath shortly before use.

5) Culture transferred to 4 15ml tubes (so only using about 60% of it, a bigger centrifuge will help us improve yield at this step), and spun at 1400rpm for about 25 mins. (slow takes a long time to pellet, ideally should be 4000rpm). 6) liquid ('supernatant') removed from pellet; pellet resuspended in 1ml ice cold MgCl2, transferred to an eppendorf and kept on ice for 30 mins. 7) Spun down in microfuge, 10mins at 6500rpm. 8) Supernatant removed, pellet resuspended in 1ml ice cold CaCl2, on ice for 30mins. 9) Spun down as in 7 10) Supernatant removed resuspended pellet in 200µl CaCl2 15% Glycerol. 11) On this attempt there was no dry ice so snap freezing wasn't possible. Aliquots transferred directly to -20°C freezer!!

Transformation:

In this case transformation followed directly after competent cell protocol using aliquots directly from step 10 above. It remains to be seen whether the other aliquots are competent or survived the (non-snap) freezing process.

1) 6µl of pUC19 DNA added to 200µl competent cell aliquot still on ice, mixed gently by pipetting, held on ice for 30 mins. Negative control is without plasmid added

note: this plasmid was from the NEB tube with cat number N3041A I could only find N3041S or L on the NEB website/catalogue. This tube says 50pg/µl which sounds like a very low conc. , if so this is only 300pg total plasmid DNA. There is a good chance I have misidentified this though!...

2) Held in 42°C water bath for 45 seconds. 3) Back on ice for 2 mins 4) Added 500µl ambient temp LB broth, mixed very gently by flicking the tube 5) 37°C incubator for one hour 6) 3x200µl of plated on to LB amp plates, incubated overnight 37°C.

Replating Wed 4 sept 2013

• Replated colonies from DH5α plate on one amp plate, DH5alpha; colonies transformed with PUC19 on another. Incubated at 37C overnight.