Hi all,
I tried to insert a restriction site in a 6.5 kb plasmid having only six endogenous restriction sites in the MCS. I already tried several primer setups but all failed. What could I possibly do to optimize the PCR reaction a get the desired PCR product. I performed PCR with Taq DNA Pol and proof-reading Vent already but it never worked. I run a PCR with random primers to check on reagents and the plasmid and this worked. Nevertheless, site-directed mutagenesis and amplification of the entire plasmid fails. DoI have to double PCR reagents for the entire amplification or add anything particular to facilitate the rxn?
Happy for any response.
Thanks in advance, Stefan
Hi, Stefan. First, i think you have to really make sure whether your designed mutagenic primers that have been used for PCR Site directed mutagenesis are already suitable or not, because the most of troubleshooting for pcr mutagenesis is on the primers since it is the critical part that determine the successful DNA amplification into the target plasmid.
If I understand correctly, your problem is that you do not get an amplicon? In the past, I found Vent to work well, but not Taq or DeepVent for classical site directed mutagenesis approaches (has to do with strand displacement ability of enzyme, I think). Colleagues and I also found that the thermocycler used had a large effect the amplification (an older machine with a slow ramp speed seemed to work best).
You may consider trying a FastCloning recombination-based approach (Li et al. 2011 BMC Biotechnol; PMID: 21992524). I've extensively used the technique for plasmid manipulations (site directed mutagenesis, deletions of segments, introduction of ORFs etc) and had great success, including SDM on large plasmids (10kb). In short, you would perform an inverse-PCR using primers with tails that introduce your restriction site (use Phusion), remove the template with DpnI, then transform this into E. coli (which recombines the linear amplicon into a circular, replicating plasmid). The size of your plasmid makes me guess that the amplification stage will likely be improved by linearizing it prior to amplification (using a site in the MCS next to where you insert your new site; will allow ends to freely rotate and thus avoid strain due to supercoiling).
Depending on the intended downstream applications of your modified plasmid, you may be able to skip the introduction of the restriction site altogether. Simply recombine your gene/insert into the vector instead - or introduce the restriction sites together with the insert if you want to excise it later on.
Hi Stefan
I work with a plasmid of similar size but instead of Taq, I used Pfu polymerase and it work well for many site-directed mutagenesis of 1-3 nucleotides.
Using Pfu allows to use higher temperatures for PCR.
@Alexander B Westbye: I use primers where the restriction site is in 5´overhang. I think it´s a good idea to first linearize the plasmid to eliminate any supercoiling e.g. with NaeI (the site to be mutagenized; creating blunt ends). I will gel extract, dephosphorylate the plasmid and then run the PCR with those primers and Vent DNA Pol for proof-reading issues. Do you think this will work?
Hi Stefan. In classical "quick change" SDMs, the mutations should be in the center of the primer - however, it sounds like you have your sites in a 5' tail? Since you're incorporating a restriction site, a cloning approach should work well.
Based on my understanding of your primers, I would skip gelextraction and dephosphorylation if you use DpnI. Linearize plasmid with NaeI, run inverse-PCR (~18 cycles). If you have a decent product then go ahead: Remove the original plasmid using DpnI digestion (it will shred the original methylated plasmid, assuming you use a standard host, but not touch the PCR-amplicon). I would then cut using the restriction sites incorporated in the primer tails (assuming a full site is present in each tail), followed by ligation and transformation. (If you don't have DpnI, you could cut with NaeI a second time _after_ the ligation to avoid transformation of re-circularized orginial plasmid)
Hi Stephan,
I have introduced restrictiion sites into large cojnstructs (~14kb) using the Q5® Site-Directed Mutagenesis Kit from New England Biolabs. These come with optimised DH5-alpha cells that should be able to handle 10 kb comfortably. If necessary you may transform into Agilent Technologies XL10-Gold Ultracompetent Cells instead of the prprietory DH5-alpa cells. These cells are ideal for extremely demanding cloning, large plasmids and plasmid libraries. Efficiency: ≥5 x 109 transformants/µg pUC18 DNA Contains.
It is imperative to deisign the primers according to the kits recommendation and optimise PCR conditions accordingly.
This has always worked for me
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