I have done site directed mutagensis by designing the primers at the mutation site, and whole plasmid amplification was performed using Hifidelity enzyme XT-20 (which can amplify up to 20 kb). The size of the whole plasmid here is 7 kb. After I got the PCR product. I have done the parental digestion using DPN1 (fast digestion for 10 minutes at 37 deg) and run both of them on the gel (picture provided- just beside the marker is after DPN1 digestion and right extreme is just after PCR amplification). I also have measured the amount of DNA which is around 52 ng/micrlitr. And I used 50 ng, 100 ng, 150 ng and 200 ng of DNA. But still I am not getting the transformation. Should I go for ligation step after this? Any suggestion would be appreciated.
Which strategy did you use to design the primers? Stratagene's QuikChange or another protocol?
If you only have a linear PCR product, transformation efficiency is extremely low compared to circular plasmids. You can self-ligate your plasmid either by using 5'-phosphorylated primers or phoshorylating your purified PCR product followed by a regular ligation.
If you want to boost efficiency go for ultra-competent cells e.g. XL10-GOLD. I sometimes have mixed results with QC. I often repeat, perhaps using an aliquot of DpnI that I know works (it can go off) and this commonly gives me better results.
You mean that you have no colonies, or wild type colonies? I used protocol where I have two PCR steps, you first make few pcr rounds with one primer and then you stop and add second primer. This avoids self annealing of primers and give better yealds of PCR product. Also you should add very litle of the template. I can send you mentioned protocol.
I use usually the Stratagene QuickChange protocol for primer design and PCR on the circular plasmid. I grow the plasmids before in DH5alpha and after the PCR digest DpnI for 1 hour. Critical point is that you use a methylating strain and PCR on the circular plasmid. Transformation works best in XL10 Blue or Gold.
How are are you that you have circular DNA? When running it on the gel, are the bands running at 7kb? Circular DNA run lower than linear DNA. Check the size of your bands to see whether they are circular or linear DNA. What transfection protocol are you using and what cells are you trying to transform into? For example, in term of bacteria, it is much more difficult to transform Pseudomonas compared to E. coli using electroporation and requires different transforming condition. Check to make sure that your protocol is optimize for your cells. Good luck.
Hi ! Maximilian
How can I simply phosphorylate the end, can you please suggest some protocol for this, which could be workable in my case ?
Thank you
Hello ! Anna,
Yeah as I have mentioned that there is no single colony. But here I get the amplified product. I have added approx 10 ng gm of for the pcr. So I thing linear product may be the problem. The transformation was done in DH5 alpha strain, and the positive control gives confluent growth but not with the pcr product. It means there is no problem in transformation but in DNA topology. so for linear or nicked DNA problems are there.
Hello Prakash,
Try to do DpnI treatment for 1h at 37 degrees. I normally take 10ul for transformation and I use XLI-Blue. After transformation I centrifuge cells for 2min at 2500rcf, decant the supernatant and leave 100ul in the tube to resuspend the cell pellet and streak it on the appropriate plates. Hope it works..
For the protocol I would suggest the following (it worked for me, with some ancient T4 kinase, not great but I got some colonies):
- Do a PCR with your primers, 4x50 microl, to get plenty of product
- Pool and column purify it, run it on a 1% agaroseTAE gel, extract it in a minimal volume
- Set up the phosphorylation as described here (smaller total volume): https://www.neb.com/protocols/1/01/01/non-radioactive-phosphorylation-with-t4-pnk-or-pnk3-phosphatase-minus
- Transform plenty of your bacteria
Good luck!
For QuikChange protocols, you almost always need competent cells with efficiencies of 10^8 CFU/ug or better. If you are using subcloning-grade (10^6 or 10^7 CFU/ug), you will have problems recovering colonies due to the low efficiency of transformation with linear DNA.
In addition, using too much DNA in a transformation reaction is deleterious. Try 20ng or less TOTAL DNA (typically around 1-2 uL of your DpnI-digested reaction).
Finally, it is difficult to accurately size your DNA from the gel you've shown. Try a low percentage (0.7%) agarose and run for longer. If for some reason, you are only achieving partial plasmid amplification, you will not get any colonies. To be certain of this, try running linearized vector in a third lane to make sure the expected product size matches up.
Thank you Every one... !! Well I got some (3-4 colonies) colonies, those colonies are able to grow on even higer concentration of Antibiotic (selection marker) and I also isolated the plasmid from there, hope the transformation is succesfull. I am sending this for sequencing.
Hello Prakash, i'm having the same problem you posted here (some years ago) related with transformation after PCR quich change amplification protocol, would you please tell me what did you do to overcome this issue!!??
thank you in advance
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