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Impossibly small ligation products


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#1 AndrewPage

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Posted 07 September 2004 - 11:42 AM

Here's a wacky one: I am ligating in a 4KB insert into a 6KB vector (I am having to Klenow both and blunt clone). I am treating the vector with CIP to avoid self-ligation. After transformation into E.coli I get plenty of colonies, but when I extract plasmids they are all around 2.25KB (too small to even be the vector on its own).

I don't believe I am contaminating with another plasmid.

I thought it might be a cryptic rearrangement of the DNA, but someone else in our lab just got similar results (ended up with more like 2.0KB plasmids) using a different vector (5KB) and insert (2KB).

Any ideas anybody?
Cheers,

Andrew.

#2 blasko

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Posted 13 September 2004 - 05:28 AM

Perhaps, you over treated with Klenow, and it has a 3'-5' exonuclease activity, and digested your vector.. (you should ask your collage in your lab, -you mentioned there are several similar results- that (s)he tried to generate blunt end by Klenow...)

when you digest by Klenow, use VERY little amount....




B.

#3 iamfat

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Posted 13 September 2004 - 07:01 AM

how did you purify the vector?
cut gel after digestion?
does the band positions are correct?

#4 AndrewPage

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Posted 13 September 2004 - 07:26 AM

Thanks for the replies.

I extracted plasmid using a fairly standard alkaline lysis protocol with isopropanol precipitation. I ran some on a spec and got a good yield with low 230 and 280 readings.

I purified from the gel (0.8% Seakem GTG, 5v/cm, 1hr) using an Eppendorf Perfectprep kit (but I have also tried the Zymoclean kit from Zymogen and a homemade column). I ran products of this and got good bands (size and intensity).

I used the Klenow from NEB (Cat # 0210). I diluted to get the recommended concentrations of everything (diluted enzyme in 1x buffer), and ran the reaction for 15mins as specified in the instructions. I skipped the heat inactivation because I did a phenol:chloroform cleanup and precipitated again to be sure I didn't have any salt or enzyme carryover. I ran some of this on a gel and got a pretty good yield.

The other cloning that has given strange results in the lab did use Klenow.

I am currently religating the vector without the old insert, so that at least I won't need the gel to clean it up after digestion in the future. I might even be able to find a blunt cutter to use on it if this works, which would be one less thing to go wrong...

So you think it's the Klenow? Do you use less than the manufacturer's say?

Thanks again,

Andrew.




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