Is the vector leading the problem?
John R. McQuiston
zje8 at cdc.gov
Mon Feb 1 08:44:54 EST 1999
Instead of blunt ending and ligating together which is a little much to ask of
the enzymes especially in a 12 ul reaction (glycerol ?) try cutting first with
XbaI and then filling in, heat inactivate/cleanup and then proceed with the
rest of the procedure. This should help with the last step. Assuming that
your enzymes are in 50% glycerol, as is common, you are pushing the limits of
the conc. in the final reaction. You can also use a larger volume, precip. then
transform. Also, I like 14C overnight for ligations. 4-8C seems a little cold
but I've never tried it so I don't know about that one.
When you ligate the vector only are you getting any colonies? This would
indicate incomplete double digest. I use a + and - ligase control with a
double cut vector to assess the ratio of double cut to single cut vector.
Another approach would be using PCR and incorporating better sites into the
In article <126.96.36.19990201210816.00683bc8 at www.kali.com.cn>,
wenfeng.chen at WWW.KALI.COM.CN says...
>We are cloning genes which are 380 and 800 bp respectively. They
>were obtained by double-enzyme cutting with XbaI and NotI (both
>sticky ends). The vector is 9.9 Kb and treated by double-enzyme
>cutting with MscI(blunt end) and NotI. following is the detail
> |(ligate the NotI ends:)
> MscI/NotI |
>Vector 9.9Kb (115 fmole) ---------->| add 1 unit of ligase
> |\ligation (25C,2h)
> | ----------------->
> |/in 10 microliter
>Insert 380/800bp(625 fmole) ---------->|
> XbaI/NotI |
> |(ligate the blunt ends:)
> | (MscI and XbaI)
> | add:
> | 3 Unit ligase \ in 12 microliter
> | 1 Unit klenow enzyme ------------------>1.5 microliter to
> | 100 microM dNTP / 4-8 C, over night
>transfect E. coli. No clone can be obtained. The ligase has
>been identified working well. What is the problem? Any suggestion
>of it is highly appreciated.
>Thank you very much!
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