Scaling up PCR to get more DNA
Posted 22 June 2013 - 02:12 PM
Unfortunately for our transcription procedures we need ALL of this DNA for 1 reaction, which means we have to keep re-doing PCR to get more DNA.
Does anyone know, or can lead me in the right direction as to a procedure to scale up PCR, maybe to around 1mL? Right now we use PCR tubes that have a max volume of 200ul. Of course we can just scale up to 200ul reactions, but is it safe to assume that a 4x scale up would result in 4x more product? Would the concentrations of Buffer, dNTPs, and enzyme be proportional? What about the Denaturation, Annealing and Extension times?
Also, if anyone is wondering we started out trying to go through bacteria cultures, but the plasmids resulting for these cultures all have mutations in the same regions, which led us to assume that maybe the DNA is toxic to E. Coli cells.
Also, I am aware that we can do like 96 50ul reactions to obtain more DNA, but I'm hoping there is a more efficient procedure.
Any guidance would be appreciated.
Posted 22 June 2013 - 02:32 PM
96 50 ul reactions isn't all that inefficient, just make a mastermix so that you have it all in one tube to start with, use 96 well plates, pool the reactions afterward, simple.
Posted 23 June 2013 - 12:14 PM
Hmm I figured there would be a problem with finding a machine, and the whole heating situation...I guess I can go ahead and do a couple tubes per cycle instead.
Posted 23 June 2013 - 12:44 PM
Posted 24 June 2013 - 07:20 AM
Another idea for larger tubes would be the very old way with water baths of different temperatures , here you can submerge the tube at least and so have a better contact...but I'd do it as last resort and a technician at hand
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.
Posted 26 June 2013 - 05:28 AM
Just ligate the (Taq polymerase) PCR product to the vector and transform into E. coli. Make one Maxi prep to yield some micrograms of plasmid, then cut it with your restriction enzymes and do gel extraction.