Problem with directed mutagensis - (Nov/29/2006 )

Hi

I have doing site directed mutagensis for last 3 weeks. and may be making the same mistake again and agian. that I am not doing the dpn1 digestion properly..cause I find lots of colonies after transformation and the colonies do not show any mutation. I was doing purification after PCR and then digestion and then again purification then transformation.. But Now I realized, I was not using the buffer for dpn 1 digestion.. Am I right???????

Please help me out as soon as possible. My supervisor will kill me.

yup, you're probably right.
do you run a gel after the PCR, and have a visible product? i know many of the protocols say go ahead even if you don't see a product, but it's usually not a good idea.
do you run a gel after DpnI digestion, and have a control? you should be able to see if you're enzyme is working.

V

Argh, this mutagenesis thing is a tricky thing in ALL of its steps. I have been trying this for quite a while now.

But I've checked the DpnI digest with the Rxn buffer that comes along with the kit and with its "own" buffer and it made no difference in performance.

What you need to make sure of is: your DNA needs to be at least 80% supercoiled,do both controls at least once just to check(the pUC for transformation efficiency of the cells you're using, the Whitescript to control the mutagenesis reaction), try to transform different amounts, check the amount of antibiotics on the plates is right for your plasmid.

But still, the mutagenesis is tricky and I'm not convinced of ever finding the mistake in it, but hopefully a little Voodo and lots of chocolate will do the trick!!!

hm,

in my hands it works quite fine. I do the dpnI digestion directly in the pcr tube (take an aliquot first for the gel) overnight. Then i take another aliquot and transform everything into the cells. I then check the aliquots (both should be the same size).

usually I get a lot of clones, mainly positives

When I do mutagenesis, I do the following: perform the PCR for 40 cycles, to make sure it works. Next to it, on the gel, I load an equivalent dilution of the original plasmid, to make sure that it is PCR-product I'm seeing, and not the plasmid itself (which should never be the case, as you add small amount to the PCR, but loading it on a gel doesn't hurt either).
When I see the PCR works, I go ahead, re-do it but now for only 18 cycles, then I add about 10 Units of DpnI to the PCR itself and have it @ 37°C for maybe 2 hours (10 units should be able to cut 10 µg of DNA in one hour, and I of course only add nanograms of DNA to the PCR, talking about overkill...). After this, I purify the product (Qiagen PCR purification) and I proceed as normal.

Works perfect.

I buy enzymes, competent cells separately, makes things cheaper mostly.

I suggested to take a sample before DpnI digestion and one before transformation, to check on a agarose gel, if you still have DNA.

Sequencing can be quite expensive. So think about redoing the SDM.


About your holidays .. I dunno