Site-directed Mutagenesis - (Jun/28/2006 )
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right, I have not checked the DNA yet by sequencing. I meant I got colonies for one sample only.
Because I precipitated the DNA before I did the digest, I diluted the whole DNA I got with water in the amount which is needed for a digest. A standard digest in my case would be (20µl total volume): 2µl buffer, 1µl Enzyme leaves 17µl DNA+water to make up 20µl total volume. Got it???![wink.gif]()
Instead of trying to transform I would check the 20 colonies that you've already got, you mght have a clone in there which already does the job.
QUOTE (vairus @ Nov 2 2006, 09:40 PM)
I've used PfuUltra for SDM and it worked like a charm, so I doubt that would be the problem.
What do you mean with "one mutated plasmid"? Is it a plasmid with your desired mutation, or with an extra mutation elsewhere or only with a mutation elsewhere?
Something that has worked for me doing SDM on quite large plasmid was to do the PCR and the digestion (more DpnI and 2 hours longer than stratagene's protocol, just to make sure), then purify it (column based, elution in 50 µl of water) and I vacuümed up my reaction to about 20 µl, then ligated it (about 10 µl of the eluate in a 20 µl reaction) transformed 5 µl of it, got a lot of colony's... (transforming Dh5alpha, no special bacteria whatsoever)
What do you mean with "one mutated plasmid"? Is it a plasmid with your desired mutation, or with an extra mutation elsewhere or only with a mutation elsewhere?
Something that has worked for me doing SDM on quite large plasmid was to do the PCR and the digestion (more DpnI and 2 hours longer than stratagene's protocol, just to make sure), then purify it (column based, elution in 50 µl of water) and I vacuümed up my reaction to about 20 µl, then ligated it (about 10 µl of the eluate in a 20 µl reaction) transformed 5 µl of it, got a lot of colony's... (transforming Dh5alpha, no special bacteria whatsoever)
right, I have not checked the DNA yet by sequencing. I meant I got colonies for one sample only.
-deuti-
QUOTE (deuti @ Nov 3 2006, 05:13 AM)
what do you mean by taking up in amount of water needed for standard 20ul digest?
got 20 colonies for one sample.
got 20 colonies for one sample.
Because I precipitated the DNA before I did the digest, I diluted the whole DNA I got with water in the amount which is needed for a digest. A standard digest in my case would be (20µl total volume): 2µl buffer, 1µl Enzyme leaves 17µl DNA+water to make up 20µl total volume. Got it???
Instead of trying to transform I would check the 20 colonies that you've already got, you mght have a clone in there which already does the job.
-britzelbeere-
Pages: 1