Few colonies with cloning of PCR product after digestion - (Aug/19/2006 )
I have a problem with my PCR products. Actually, I do not know if it comes from that but when I transform the competente cells with the PCR products I do not get any colonies (alomost nothing) or at least I get only some on my positiv control (which is made with10ng of DNA template). I am using the Quick changed Site directed mutagenesis kit with Dfu turbo and with a 10xreaction buffer MgCl2.
I have tried to run a gel (before adding the restriction enzyme DpnI) to check the amplification by PCR was ok and it was the case indeed, I could see the bands.
So, I do not know why I get only a few colonies on my plates (and actually I think that it looks more like contamination ). An other thing: I have not used a buffer when I added DpnI, maybe it is the reason??)
If you have any suggestion, do not hesitate.
I don't use a buffer when I add DpnI neither. (if the DpnI wouldn't work, you would have more transformation, but not less)
how much of the PCR product do you use to transform your bacteria?
(I use only 1µL of PCR product (out of 50 µL) for 100 µL of competent bacteria. I am not sure if too much PCR product can inhibit the transformation?)
Thanks for your reply, actually I use 10µL out of 50µL of PCR product for 100µL of cells. I think the amount is all right. I will try to found out what is wrong .
Adding too much DNA + buffer to the competent cells can indeed cause problems. We find anything more than 5% quite inhibitory. Listen to Missele.
Ok, I will try to use less PCR product. Concerning the buffer for DpnI I think I should use it because it was included when I ordered the restriction enzyme. And I suppose that my problem must happening after the addition of DpnI since I can see the bands of the PCR products before adding it. I am going to run a gel after adding DpnI as well, just to be sure.
OK, add the buffer, it will not hurt, unless you add finally too much salts (don"t forget that your sample is already buffered by the PCR buffer. the only thing that could miss is MgCl2 and DTT. check your PCR buffer). But I still claim that it is not a problem of DpnI. your problem is somewhere else
Let's check the different steps :
the PCR. You say that the amplification is OK. I assume you loaded on the gel the same amount of DNA template that you used for the PCR, to see an increase of the band after amplification.
Then it's not a problem of PCR.
The DpnI digestion.
The aim is to digest the parental strain, (the template of PCR, the methylated DNA).
What would happen if this step would not have worked? you would transform bacteria with the non-mutated plasmid. you would get transformation of the wrong plasmids, and not an inhibition of transformation.
Your problem is the transformation.
Thanks for the advices Missele, well I am testing the few colonies I got in order to see if they contain the mutations. Otherwise if it is not ok, I will have to find what is wrong with the transformation.