Side directed mutagenesis with 17kb plasmid - (Dec/11/2005 )
This will decrease your accuracy. And with site directed mutagenesis you want to have the smallest amount of extra mutations as possible. Also, I'm not sure if TAQ has strand displacement activity, which pfu doesn't have @ 68°C (not having strand displacement is necessary for mutagenesis).
You might want to try the Phusion enzyme from Finnzyme/NEB. It has high processivity and good performance on long templates.
Hi
I have done SDM with a 6kb plasmid and followed the stratagene protcol, except I used 20 units of DpnI for digestion. before that I could see the bands on the gel.But after transforming them into competent cells, I did not get any colony. I changed the transformation protocol and then found 20 colonies on one plate. But others do not show any colony. Is the problem in transformation? Do I need to use super competent cells?
Help me out
Dear All,
I have problem as well with SDM.I am trying to insert one nucleotide mutation.
It seems that PCR protocol worked (with 17min elongation and PFU)...Now, the size of the mutated plasmid is about 13kb and another about 10.5kb. As I said after PCR I check on gel and seems to work. When I perform transformation (10ul Dna(previously treated with DpnI) in 55ul XLbluesupercompetent) I am not able to see any colonie..
Any idea..thank you in advance...
I would decrease the volume of DNA added to the cells. You can precipitate and decrease the volume, so that you would actually use more DNA for transformation.
Apart from this, have you checked the competence of your cells?
Probably..I will check but I think the competences is ok, furthermore I tried once with cells coming from the kit and the results was "no colonies" as well.
What if I increase the volume of cells up to 200ul + 10ul DNA? I found an interesting:) protocol for transformation that suggest to use more cells...
I treated DNA with DpnI and then I purified it with pcr purification kit. Then I transformed (100ulcells+5ulDNA) and I got colonies ..incredible!! just for one mutant actually, but the other si really big.. I will let you know my progress.
Thank everybody
I have problem as well with SDM.I am trying to insert one nucleotide mutation.
It seems that PCR protocol worked (with 17min elongation and PFU)...Now, the size of the mutated plasmid is about 13kb and another about 10.5kb. As I said after PCR I check on gel and seems to work. When I perform transformation (10ul Dna(previously treated with DpnI) in 55ul XLbluesupercompetent) I am not able to see any colonie..
Any idea..thank you in advance...
Hi CSI,
As iwas abroad, I could not reply you in correct time. What I did was, I added almost 10 ul of PCR purified DNA into 40 ul competent cell and kept on ice for 45 mins, When I was adding the DNA, I also swirled the epp tube on ice.After 45 min, I gave heat shock at 42 degree for 45 min in waterbatch. remember this time limits is very important and do not shake while giving heat. then i kept the tube on ice for 2 min and then transferred the DNA to 14ml falcontubes and incubated at 37 degree with 160 rpm shaking for 1 hour. then i spreded the DNA on preaheated ( 37 degree0LB selection plates and incubated in 37 degree for overnight.
hope it would work.
Deuti
Is it possible to do site directed mutagenesis just by one primer?Im cloning a gene in puc18 after that im gonna change an amino acid to another just by using one primer. Is it possible??
I am working on a side-directed mutagenesis with the Stratagene´s quickchange mutagenesis xl-kit. Here is my problem: My plasmid is 17kb big. Until now I have had no colonies on my plate. According to Stratagene´s protocol, I used the following cycling parameters:
1. 95°C 1 min
2. 95°C 50 sec
3. 60°C 50 sec
4. 68°C 1min/kb plasmid (so for my plasmid 17min)
5. 68°C 7min
As I have already said no colonies! Then I read that the Pfu-polymerase needs 2min/kb of the plasmid in the elongation time. So I increased the elongation time from 17 min to 34 min. But again no success. I would like to ask if anyone has got an idea.
Thanks in advance!!!
I think that it could be a couple of things. First, what kind of cells are you transforming into, and how long are you incubating the plates? Some supercompetent cell lines are very fussy, lose their competence after a few thaw/refreeze cycles, and take about a day and a half to grow colonies. You may just have a poor transformation efficiency. Second, I'd shorten the elongation time to 1 min. to just PCR a fragment. Run it on a gel, if you dont see a band for the fragment then your primer is either bad or not annealing properly. If this is the case, try adding 3% DMSO before PCR. Third, the polymerase could be bad as well. Order new Pfu. Another thing that it could be is your program. Try this four-step PCR:
1. 95 30sec
2. 95 1min
3. 55 1min
4. 68 1min/kb cycle 20 times to step 2