Help???How to cip treatment with blunt-end vector???

Chris Fields cjfields at jove.acs.unt.edu
Wed Aug 27 20:53:39 EST 1997


EcoRI, a BLUNT-END cutter...hmm, I missed some meeting (the original poster
was talking about blunt-end ligation, not sticky-end).  If one read Maniatis
a little more, then one would realize EcoRI was a sticky-ended cutter... oh,
I'm sorry, I forgot, Maniatis is old and unnecessary, according to you, dear
sir.  Let me know when you come out with your own three-volume set on
molecular cloning so I remember not to buy it.

Sorry to say, I guess I'm one of the few people who'll admit to using
Maniatis's "time-consuming" tomes, and I found them to be quite helpful.
Don't call something "old" when it's methods have withstood the test of
time.  And since when does a book published eight years ago make it an
antique?

You got your clone in one shot?  Congratulations!  I'll bet that this little
"trick" doesn't work every time, however.  Good luck, then, esp. if you're
not going to use Maniatis or Mr. Red Book.

I think I'll still stick to Maniatis's and my own "time-consuming" methods.


--
C. J. Fields
Graduate Student, Dept. of Biological Sciences
The University of North Texas
Denton, TX

email : cjfields at jove.acs.unt.edu
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C.Rodemer wrote in article ...

>In article <33F4A548.5D90 at chugaibio.com>, dspinella at chugaibio.com,
>@chugaibio.com wrote:
>
>> > Does someone know how to CIP treatment with blunt-end vector???
>> > many thanks,
>
>> This is pretty basic stuff, Dong.  You need to get your hands on a
>> recipe book like Maniatis or the Red Book if you're going to fool with
>> stuff like this, else you're just going to get in over your head real
>> fast.
>
>I think many things in the Maniatis book are unnecessary,old and time
>-consuming.
>
>>  Anyway to answer your question, precipitate your vector with
>> ethanol, resuspend in Cip buffer (1mM ZnCl2, 1 mM MgCl2, 10 mM Tris-HCl,
>
>I cut my PBluescript with EcoRI and didnt precipitate anything.I just
>added buffer for CIP and some Units CIP .Then incubate at 37C.
>
>> pH 8.3) and add 1 U of CIP per pmole of blunt-ended linear vector.
>> Incubate reaction 30 minutes at 37 degrees (or just add Cip directly
>> your restriction digested DNA).  Kill the enzyme by adding SDS to 0.5%
>> and EDTA to 5 mM and Proeinase K to 100 ug/ml.  Incubate 30 minutes at
>> 56 degrees.  Cool the reaction to RT and phenol/chloroform extract and
>> then precipitate.
>
>Well, I just made two phenol/chlorof/IAA extractions and precipitated  the
>vector with 2 vol ethanol. Then I used it for ligation.
>Obviously it worked. I screened only 10 cols and I found the recombinant
>pBS in one colony.
>
>bye
>C.R.
>
>--
>To send me Email, remove .nospam from my address.





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