fill-in and blunt-end ligation

David Micklem drm21 at mole.bio.cam.ac.uk
Wed May 14 06:32:10 EST 1997


In article <33795a3f.1164464 at news.univie.ac.at>, a8803349 at unet.univie.ac.at
(Martin Offterdinger) wrote:

>On 12 May 1997 22:29:18 GMT, Mari.L.Shinohara at dartmouth.edu (Mari L.
>Shinohara) wrote:
>
>>I have a hard time cloning.  Unfortunately both fragments, insert and
>>vector, did not have good restriction sites and I had to cut them with
>>all different enzymes generating  5'-protruding ends.  Then I
>>dephosphrylated only the vector fragment first, which has the Amp gene
>>there (I don't think it matters whether I dephospharylate this before
>>or after filling-in), and filled both fragments with Klenow with dNTPs.
>> Blunt-end (supposed to be) ligation was performed finally at room
>>temperature for 2 hours with T4 ligase.  --- I tried this more than 5
>>time, but it really doesn't work...
>>
>>Does anyone have idea why I can't get it?  Or if you have any advice,
>>it would be greatly appreciated.
>>
>>Thank you in advance.
>>
>>Mari
>
>1) It is very important to remove ALL alkaline phosphatase (normally
>calf intestine AP) before ligating, with two phenol extractions.

Or gel purify...
>
>2)Dephosphorilation should be performed after filling in!

Hmmm, why?  Its a 5'-phosphate, so the fill-in reaction won't affect it.


Other recommendations: _don't_ use excess phosphatase!.

However, you really haven't provided enough information! What doesn't work
-too many colonies on the negative control? No colonies at all? All empty
vector? How did you clean up the  fragments? 

Another tip: depending on exactly what enzymes you used to cut your vector,
and what sites are in your insert, you can sometimes re-cut your plasmid
AFTER ligation in order to cut down on background.  For example, if you
blunt-end clone into an EcoRV site, and the product you WANT doesn't have
an EcoRV site, then you could cut the ligation mixture with EcoRV (just add
it with the ligase) to re-open recircularised vector.

David

-- 
D.R.Micklem,                                Time flies like an arrow... 
Wellcome/CRC Institute,            Fruit flies like a banana.       
Cambridge CB2 1QR, UK              
Email:drm21 at mole.bio.cam.ac.uk Junk mail very very unwelcome.



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