PCR off midiprep - problem with downstream reactions? (Nov/01/2005 )
I am trying to use PCR mutagenesis to delete pieces of my vector for interaction studies and am having problems. I have tried changing most of the variables (annealing T, Mg2+, primer and template concentrations) and it still isn't working. A colleague of mine has suggested that it may be a problem with the template DNA. I am working with a midiprep done using a kit from Sigma Aldrich, which uses alkaline lysis and a silica column.
Has anyone ever had problems PCRing off this type of template. What about other purification methods and downstream PCR?
Any advice or anecdotes would be helpful,
i have succesfully done PCR on alkaline-lysis midipreps.
I think that it can also be a problem of polymerase. I've sometimes been unable for several assays to PCR a fragment with one pol, and had success on the first try woth an other one...
Are you 100% sure you have the DNA (checked it on gel, or spectrometer)?
If you aren't succesful, EtOH or isoprop. precipitate it maybe?
The most likely problem is that you are using too much plasmid DNA in your PCR reaction (Too much plasmid DNA will kill your exponential amplification) Try doing the PCR using a dilution series of the plasmid (say from 1:1 down to 1:1,000,00) and see if that works.
Hello Everyone and thanks for all the replies!-
I am sure the DNA is in the tube at at least around the value I received from the spectrophotometer as I have run it on gel several times.
I suppose I could try another enzyme, but for this type of cloning project, I generally use a 1:10 ratio of PFU Ultra to normal Taq (the PFU can proofread the taq and it's economical), so I've actually tried this with two different polymerases (sort of).
How does too much DNA kill the amplification of the reaction? In my setup I use 25ng as a start but only do 17 cycles as I'm PCRing around the entire plasmid, to limit errors incorporated by the enzyme. I then treat with DpnI to kill parental (methylated) plasmid and transform. I've heard of others using a similar amount of starting material for this type of amplification.
I just finished trying to PCR off another template to see if for instance I oxidized the DNA by lysing in NaoH/SDS for too long during the midiprep, but I'm usually pretty careful so I'm not exactly holding my breath for this one to work. I'll post when once I find out the result.
Thanks for the Help ,
PS I'm kinda getting to the end of things to try as I've already tried changing the primer, Mg2+, and template concentrations independently and now I'm trying the template. If this doesn't work I a was thinking about using another primer I've got in the freezer to PCR out just my insert, but the problem is that the melting temperatures for the two primers are about 20 degrees C apart.
Is this worth trying? I'm not super experienced with PCR. I never really understood why exactly the Tm's had to be so close together.
do a positive control with different amounts of template ranging from 5 to 30 ng and check about the appropriate one; my exp is that 10 is enough and i decrease the amplification when starting with more than 25.
i've tried to amplif a sequence with Taq, then Pfu, then phusion polymerases. None worked. I've got success with eppendorf triple master, wich is believed in my lab as one of the more able to amplify.
But a colleagu who try to amplify big insert 7kb doeasn't had success with enzymes in our lab + from neighbour one...
Finally, two primers by annealing different from 20°c, i would try for sure...
good luck, and keep posting.