Is the vector leading the problem?

John R. McQuiston zje8 at
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 

Good Luck.


In article < at>, 
wenfeng.chen at WWW.KALI.COM.CN says...
>Hi, there,
>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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