Site-directed Mutagenesis - (Jun/28/2006 )
QUOTE (britzelbeere @ Nov 3 2006, 01:41 AM)
What worked for me was the PCR with 68°C 2min/kb plasmid, purify and take up in amount of water needed for standard 20µl digest, then stop reaction and purify and then transform 2-6 µl (out of 50µl). With this I got nice colonies on my plates. All other attempts just failed.
what do you mean by taking up in amount of water needed for standard 20ul digest?
I did PCR, added DpNI 20units to the 50ul PCR product and then took 5ul of digest, transformed into 50 ul competent cell using the heat shock method.( 45 min ice, heat pulse 45 secs, ice 2 minutes, added 200ul LB, incubate ON at 37 degree) and found no colonies for three samples, got 20 colonies for one dample.I tranformed again in super competent cell, this time no colonies!!!!
-deuti-
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-