Scaling up PCR to get more DNA - (Jun/22/2013 )
Right now we are doing 50ul PCR reactions, which after gel extraction, RE digestion and PCR clean up yields about 400ng of DNA.
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.
The difficulty with scaling up to 1 ml will be finding a PCR machine that can take 1 ml tubes... unless you do it by hand. Also, in my experience, scaling works to a point, and in theory should give you proportional yield, but in practice, anything over 100 ul is difficult due to uneven heating of the tube (think about how much of the tube is actually in contact with the Peltier block).
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.
I'm not sure what you mean by 96 well plates? How would that fit into a scheme in which the PCR machine holds PCR tubes?
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.
You can get 96 well plates and 8 well strip tubes for PCR machines...
The older thermal cyclers often have heating plates for 0.5 mL tubes and were slow with heating and cooling rates. This means if you scale up with a larger volume, IMO you should mostly increase the times a bit so that the liquid has more time to heat up or cool down...But this makes it more susceptible to unspecific reactions at unwanted temperatures. So the primers should be good and specific enough and the reaction stable that works even at not optimal conditions.
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
Another possible way might be to use the PCR template for ligation into a simple vector system (like PGEM-T).
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.