I have been trying to do a site directed mutagenesis on a plasmid.I have been using the protocol for the PfuUltra II fusion HS DNA polymerase. From the gel it looks like the PCR as a success with the band at the correct position. I have taken 2ul of this product and tried to transform it into XL-10 gold competent cells, but after plating it and keeping it overnight, I find no colonies. Could anyone suggest a possible reason for this and an alternative that might work?
Try diluting your plasmid 2-fold, 4-fold. Sometimes too much plasmid inhibits transformation. Did you ligate? For some protocols, phosphorylated primers are needed so that their products are ligated later after the DpnI digestion.
check your cells, plates(is it right antibiotic?) and transformation procedure
- normally, background colonies should always appear, regardless of
the PCR success.
After digestion of the wild template with DpnI, I usually use 5 ul for transformation and keep them in SOC medium shaking at 37 oC for at least 2hrs. It almost always works.
When not, purification of the DNA product before transformation might help.
i did control transformation so that worked and have used newer cell stocks and plates. this kit typically doesnt require ligation after the digestion. i have also varied the plasmid amount...but it doesnt seem to work.
control transformation - how much plasmid did you use?
DpnI eliminates ~90% of the template, remaining template should
still give colonies.
yes so i am not sure if it is some remaining enzyme or reagent that is interfering with the transformation.
If the transformation controls worked right I would ensure the amplification is complete, despite the appearance in gel, by designing longer oligonucleotides, longer elongation times, more cycles and maybe lower annealing temperatures.
When I have problems with a difficult construct I find that it often helps to increase the concentration of my mutated plasmid. So, try doubling your PCR reaction, then after dpnI, run all of it through a column, wash, and elute with 5-10uL ddH2O. It is important that your droplet with H2O for elution hit exactly on the filter in the column (and wait app. 1 min before spinning down). Use 5 uL of the elution to your transformation. Incubate first 30 min on ice, then heat/electro transform, immediately add SOC/LB, incubate 1 hour… I usually put 200-300 uL of the transformed bacteria on a petridish… Good luck…
For me, it sometimes helps to perform a PCR cleanup (using a PCR purification kit) of the PCR product, before ligation-transformation.
thanks....i actually did that and looks like it helped. i could get it transformed. waiting for the sequencing results to confirm.
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