Why I keep getting no colonies from transformation of PCR products - help! (Nov/27/2005 )

Howdy, everybody:

In last week, I performed mutagenic PCR reactions with the stratagene site-directed mutagenesis kit, substituting two bases in the parent plasmid DNA (pTaxH6). Dpn I digestion was performed on the PCR products. To make sure if there is DNA after digestion, I ran all the PCR products together with the parent plasmid. I did see the correct size DNA on the 1% agarose gel. So, I continued to transform those DNAs into competent SCS1 cells (lab-made cells from commercial SCS1 cells, which may be not as competent as the commercial ones). In my first transformation, 7 ul of each DNA was added to 50 ul cells, which was followed by incubation on ice for 30 minutes and heat-inactivation at 42 degrees celsius for 45 seconds. The cells were then put on ice for 2 minutes, and then incubated at 37 celsius on shaker for 1 hour. 200 ul of each transformation reactions were plated on LB + Ampicillin(100ug/ml) + Chloramphenical (34ug/ml). However, this transformation yielded no colonies after overnight incubation at 37 degree Celsius.

So, I did a second transformation with slight modification of the above described one. This time, I incubated the cells on shaker at 37 degrees for 2 hours. However, again, I failed to get any clonies from the transformation.

I really don't know what was wrong with my experiments. The mutated DNA cannot transcribe the Amp and/or Chlor genes?? or the transformation efficiency is too low for the lab made SCS1 cells??

Any ideas/input will be greatly appreciated.

Thank you!!!

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What PCR enzyme are you using -- you must use an enzyme which does not strand displace, such as Pfu Ultra.

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I used Pfu Turbo which does not have strand displacement, either.

Even if there is strand displacement in the PCR reactions, there should be Amp and Chlor genes transcription of the plasmid, is that right??

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Pfu's have strand displacement @ 72°C. You must do elongation @ 68.

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In my experience it should work at either ....depends on how long your template is for the recommended. Anyway, make sure you are using the correct concentration of primers, template etc.

Make sure you start the annealing temperature at ten degrees below your Tm. You then have to vary one of the these principles. Play around just a little. Start with template concentration.

jc

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thank you for your suggestions.

I did use different amounts of DNA templates (5ng, 10ng, 25ng, and 50ng) in my experiments as well as different amounts of sodium ion (Na+). The reason for titrating Na+ concentration is that the Tm of my primers is lower than the required 78 degrees celsius. Through using appropriate Na+ concentration, I hope to increase the Tm of my primers (50 bases long). I found that the concentration of Na+ greater than 25mM kills the Pfu polymerase activity.

I also run the PCR products after DpnI digestion on a 1% agarose gel. I also saw the correct bands on the gel. Please see this image below for the DNA check after Dpn I digestion.




thanks

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