I'm try to perform site directed mutagenesis to introduce point mutation in my desired gene. So far I have failed to see any colony. I'm following in house method of liner amplification by pfu kinase, then DpnI digestion and transformation. I tried using 10 ng - 200 ng template with different concentration of primers with different PCR condition, but failed each time.
Now, after going through literature I found I ordered typical primers. My primers were not phosphorylated at 5` end and I didn't perform any ligation with T4 ligase. So, please suggest me your valuable comment about the usefulness of using 5`-phosphorylated primers in site directed mutagenesis experiment.
It depends on your specific mutagenesis protocol, the Phusion Site Directed Mutagenesis kit uses phosphorylated primers while QuikChange uses unphosphorylated primers.
The purity of the template DNA and of the primers is very important for site-directed mutagenesis. When ordering primers for this method I usually select an additional purification step (e.g. HPLC purification). This is of course more costly but usually gives good results (that is several positive colonies in most cases). Furthermore, primers should be between 25 and 45 bases in length, with a melting temperature (Tm) of ≥78°C. Also the quality of the cells you use for transformation is essential. Always use a batch that has previously demonstrated excellent transformation efficiency.
I hope this information might help you.
Best regards.
Hi there,
With the Quiagen QuickChange kit, you don't need any phosphorylated primer and you don't need to run any ligation either.
Hi,
I normally use standard non-phosphorylated desalted primers from Invitrogen and have no problems. I found really important the amount of both primers and the amount of template DNA. I am currently using KOD polymerase (Merck) as DNA polymerase with a protocol adapted to its amplification conditions originally from the Quickchange Site-directed mutagenesis Kit II (Stratagene, currently Agilent) and DpnI from NEB. For transformation I use home-made competents, XL1-blue strain.
Hope this helps.
If you are using a QuickChange Mutagenesis strategy you do not need to phosphorylate the primers. As Agata mentioned all the you need is a high fidelity polymerase (I use pFU Ultra), DpnI restriction enzyme. The main thing you are paying for on a QuickChange kit is the library grade chemically competent E. coli. However, if you use a strain like XL-1, Top-10 or DH5-alpha you can make library grade competent cells (1x10^8 or 9 cfu/ug DNA) using the Inoue method for making competent cells.
it has been almost a year since this discussion and probably you don't have issues anymore. Still, last year a paper compared different polymerases and primer overlapping sequences for optimal SDM. I recommend reading this nice work:
http://nar.oxfordjournals.org/content/early/2014/11/15/nar.gku1189.full
I agree with most of the people above: you don't need phosphorylated primers for the standard QuikChange reaction, but also HPLC purification may not be required if you can get good quality primers without purification. See for example:
http://www.nature.com/articles/srep10654
The advantage of using partially overlapping (desalted from IDT) oligos as well as Phusion polymerase and DpnI (for plasmid template digestion) both from NEB is obvious because these 2 enzymes have compatible buffers for optimal activity:
https://www.neb.com/tools-and-resources/usage-guidelines/activity-of-restriction-enzymes-in-a-q5-taq-or-phusion-pcr-mix
it is very confusion to read among different protocol of SDM from different companies including NEB, THERMO, and stratagene. stratagene seems to the simplest, but in my hand it sometimes fails. I donot know why. stratagene says just directly transform into E. COLI without the treatment of ligase and kinase, but neb needs ligase and kinase. although they need different kinds of primers, I think the resulting product is linear PCR product, so they must need enzyme to ligate the linear pcr product into a circular plasmid. however, stratatagene does not need the ligation step. very confusing!
Hai Li: Yeah, NEB creates a linear, blunt ended product without overlapping ends which needs kinase & ligase to become circularized. You don't need that for Quikchange (Stratagene) because the overlapping primers create a linear product with homologous ends that get chewed back, overlapped, and ligated together into a circular plasmid at a low frequency after being transformed into E. coli, according to the first paper Carlos G. Acevedo-Rocha linked. Obviously that can't happen with the NEB method because there's no homologous ends. That paper also suggests that the number of transformants recovered increases as more PCR product is produced, which makes sense because unless you have a specialized cloning strain of E. coli recombination of short homologous ends is a pretty rare event.
I'm guessing that many (if not most) times when the Quikchange kit fails it's because the primers weren't able to amplify the plasmid very well. Sometimes it's tricky to predict how well overlapping primers will behave.
By the way basically none of these kits do anything that you can't recreate yourself buying the enzymes separately if you ever want to trouble shoot individual components.
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