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 
insert.

Good Luck.

John






In article <3.0.32.19990201210816.00683bc8 at www.kali.com.cn>, 
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
>procedure:
>
>                                       |(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!
>
>
>Wen-Feng
>




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