king-ferdi at gmx.net
Fri Feb 17 04:00:13 EST 2006
maybe I can help you with this:
In article <1140095937.787550.64340 at z14g2000cwz.googlegroups.com>,
augcabral at bol.com.br says...
> Hello Friends,
> I am trying get a ciclic peptide by using the Impact Twin (NEB) for
> more them 3 months. So far I couldn't nor even get any viable colony
> from the transformation step. What I am doing is as follows:
> I have a plasmid with my DNA sequence of interest. I amplify this
> sequence by using PCR where I add the restriction ends for Xho I and
> Nco I.
Have you added "junk sequences" flanking your primers? restriction
endonucleases won't cut at their specific site if there are at the end
of the DNA. Different enz ymes have different requirements for the
length of the additional sequence. The specification can be looked up at
the NEB catalog or on the fermentas homepage...
> I digest my vector (PTwin2) and PCR product whith the restriction
> enzymes referred before and the condicions for that were: 37 C -
> overnight. After that, I do a dephorphorilaton of the vector with
> Shirimp Alkaline Phosphatase (SAP).
> Using the dephosphorylated digested vector and the PCR product digested
> I tried by diferents ways to do the transformation.
> The ligation has been made with T4 DNA ligase (Premega) with diferents
> times and temperature of reaction (2h room temperature (rt), 4h rt,
> Over night (on) 19C, on 8 C, on 12 C and many others. From a
> analitical agarose gel (2%) run I decide the amounts of PCR and Vector
> using the band intensity on the gel. Try keep a ratio of 1:1 PCR
> product and Vector.
The ATP in the ligation buffer often precipitates or is hydrolized after
repeated freezing and thawing. So make sure either to prepare your own
buffer and aliquot it or aliquot the bought buffer directly after
thawing it the first time.
> I have used already LB medium with ampicilin, SOC medium and I tried to
> use DMSO for the transformation.
> The transformation is made leaving the cell in Ice 30', heat shock 45s
> at 42 C, Ice again 2', add 500 ml of medium pre heated (37C) and tham
> 1h at 37C with 400rpm.
What kind of competent cells do you use? Did you buy them or did you
prepare them on your own? Did you test the competence using the PTwin
vector? What kind of positive controls did you include?
> The plates were incubated at 37 C over night.
Stupid question: Did you use the appropiate antibiotic?
> I dont know what to do because I already got 3 colonies but theirs PCR
> with the same primers I used to amplify give multi-bands. I have
> multi-bands when I also do the PCR with the primers for sequencing.
> Because that I couldn't nor even sequence anything.
> Of course I didn't get the correct plasmid but I would like knowing
> what's going on in my experiments.
> If someone has nay idea please tell me.
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