Site-directed, PCR works but no colonies - (Jun/19/2012 )

Using the Quikchange XL II kit, i've been trying to get a site directed mutagenesis to insert two 3 letter codons separated by 6 nucleotides. I did the PCR reaction and ran a gel with the pre and post dpn1 digested sample and got bands in both lanes with slightly less bright bands after the digestion. The PCR seems to have worked well but the transformation is not working at all. If i do get a few colonies, their sequences are that of the template DNA (probably an incomplete digestion). In the past when I have done this successfully, the plate will have 20+ colonies as opposed to few or none. I am lead to believe something in my transformation is going wrong.

To transform I follow the protocol using 2 ul beta mercaptoethanol followed by 2 or 5 ul of pcr product. I use a 30 second heat shock at 42C. I made new ampicillin plates (plasmid has AMPr) and still no colonies grow. I'm running out of parameters to try out. I've ordered a new kit and will try transforming with new XL 10 gold ultracompetent cells when it arrives. Anyone know of anything else that could be making this experiment fail?


Thanks!

Ph. Dizzle

PHDIZZLE on Tue Jun 19 19:50:31 2012 said:

Hi, I never used this kit for its high price, instead of using the KOD-plus from TOYOBO and the Dpn I from NEB Co Ltd. I have done almost 20 mutagenesis clones and only one constructs pick up exceeding 3 clones to gain the postivtie ones (the mutant)

I think if your procedures follow the kit, it may work straightforward.
I don't agree with the transformation problem, the transformation is so simple things to undertake.
From my experience, the PCR and Dpn I treatment is so critical for sucessful mutagenesis. PCR include the template, primer, Tm, and thermal cycles. And Dpn I treatment may add a control the template(never amplification) If this negative control grow less or no clones it may indicate your Dpn I treatment is very successful.

I got my directmutagenesis very smoothly and usually pick up 3 clones for sequencing may gain at least 1 muated clone, sometimes all are your desired ones.

I recommend you transform less Dpn I-treated PCR products ususaly 1-2 ul for 100ul competent cells is sufficient.

I've replied to in this sort problem in this Bio Forum for several times, you may collect and for a reference.

Good luck

How many cycles did you run your PCR? Did you check whether your strain from which you isolated your template DNA which you used for PCR was not Dam-?

I have never used this kit but I am a fanatic fan of quick change in case that you want to substitute just 1-2 codons like you. I am using KAPA HiFi polymerase or Pfu and then I treat the amplicon with DpnI from NEB. I have seen that prolonged periods of incubation help but what really helps is the purification of your DNA after DpnI treatment. Therefore, I encourage you to purify your DNA using a PCR clean up kit or ethanol precipitate it. Then transform only around 10-20 ng of DNA. If everything is OK up till this step, then it should be fine.

I hope that helps....

Best,
Chris