Hello,
I am trying to generate a single mutation in a 10Kb plasmid using Quick Change XL protocol. After the PCR I performed gel electrophoresis and obtained a single band. Then I performed DpnI digestion for 2h at 37°C and transformed XL 10 Gold bacteria. Everything worked fine after that, until I sequenced my DNA and found out that the mutation wasn't there. I changed DpnI, designed new primers, but nothing seems to work.
Any suggestions please?
The amount after PCR should be roughly 10-fold more than before PCR (it is a linear reaction, not a chain reaction). In our lab, before PCR the plasmid is just below the detection limit, and after PCR it is a clear visible band. When we don't see an increease, we don't bother with DpnI but first work on the PCR conditions (annealing temperature, elongation time) until we have a decent amplification.
Yes one should not use too much plasmid to start with, but also not too little, because it is a linear increase (only the original template acts as template, so at each PCR cycle you gain slightly less than the original amount on top, not more). If we had a heat stable ligase in the reaction, it could be a proper chain reaction, then we wouldn't have to bother with DpnI at all.
The "single band" you obtain after PCR must be compared with how much you have before the PCR, if it is not much stronger after the 15 cycles then your PCR didn't work. The Dpn1 digestion is never 100% complete, so you have to sequence 2-3 clones, not just one.
Two questions: Have you got the plasmid without nicks? and when you transform E.coli, Have you got many colonies?
Jurgen has a good suggestion. I have never actually tried to view the band after pcr and was under the impression it was too little DNA to see.The most likely reason your mutagenesis is not working is that you are starting with too much vector and it is not completely digested. The protocol suggests using very little plasmid.
The amount after PCR should be roughly 10-fold more than before PCR (it is a linear reaction, not a chain reaction). In our lab, before PCR the plasmid is just below the detection limit, and after PCR it is a clear visible band. When we don't see an increease, we don't bother with DpnI but first work on the PCR conditions (annealing temperature, elongation time) until we have a decent amplification.
Yes one should not use too much plasmid to start with, but also not too little, because it is a linear increase (only the original template acts as template, so at each PCR cycle you gain slightly less than the original amount on top, not more). If we had a heat stable ligase in the reaction, it could be a proper chain reaction, then we wouldn't have to bother with DpnI at all.
I did not get the linear increase part. Why is it not a chain reaction? Can you please elaborate a bit Jurgen? The newly synthesized strand is linear not circular, am I correct?
Jurgen, the protocol says to use 10ng of DNA template. Should I use less? I normally sequence 2 clones, I'll sequence more clones next time. Thank you.
I used the following NEB mutagenesis kit and the relative protocol, and it worked well:
https://www.neb.com/products/e0554-q5-site-directed-mutagenesis-kit
Sujay,
yes the newly synthesized plasmid strand is separated by two nicks from the circular template, if you draw it on paper you will see that the new linear band cannot work as template for a new PCR, hence the linear increase. However, the double nicked plasmid is quite stable because there is a 31 bp overlap or more, the hydrogen bonds of which are strong enough to keep it together until E.coli ligase repairs the nicks after transformation.
Bilal,
yes 10 ng is close to the detection limit, and since I take only a portion of the PCR reaction to check before and after PCR, it is invisible before PCR and clearly visible after PCR. 10 ng is for small plasmids (3-5 kb). If you do site-directed mutagenesis on bigger plasmids (10-15 kb), use 20-30 ng for the same number of molecules. In fact, bigger plasmids have lower copy number and transfor less efficiently, so you may have to use even as much as 50 ng. I personally prefer to do my mutagenesis in very small plasmids and subclone the relevant portion after sequencing to the final expression vector (which is usually bigger).
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