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ligation colonies contain empty vector..


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#1 kathyoc

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Posted 08 February 2012 - 05:11 AM

Hey,

I was wondering if anyone had any advice! I am trying to subclone a 4.2kb insert into 5.2 vector..... I am cutting with eco and bam.. i digested and ran on a gel and purified the insert and vector from the gel. following ligation and transformation there are no positive clones.

Now, the negative control always has colonies.. I know you can only test if the vector was single cut by each enzyme separately and assume the double worked. So I had the same vector with an insert in it already and cut that with eco and bam and I could see the insert dropout. I then took the backbone of that and one would assume that this dna had to be double digested if the insert dropped out. so why is there colonies on the negative control?

Please help!!!

#2 bithorax

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Posted 08 February 2012 - 07:14 AM

What is your negative control?
Non transformed bacteria or ones transformed with empty vector?
In the second case there are always colonies (empty vectors ligates with each-other). What helps to decrease a background is dephosphorylation of the vector (linear DNA is much more difficult to transform)..
To check presence of the insert you could try to find another pair of restriction enzymes in the vector.

#3 kathyoc

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Posted 08 February 2012 - 07:26 AM

The negative control is the double digested vector (assuming) and no insert.. Currently the ligation plates and negative control have the same number of colonies so is it even worth checking for the insert.?

#4 kathyoc

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Posted 08 February 2012 - 07:29 AM

Also, if the vector and insert are similar in size , is it more efficient to use a 1:1 ratio?

#5 bithorax

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Posted 08 February 2012 - 07:29 AM

it's up to you. me, I am lazy and probably wouldn't bother. dephosphorylate the vector after digestion - this should help.

#6 bithorax

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Posted 08 February 2012 - 07:31 AM

about the ratio I would use excess of insert, something like 3:1 or 5:1.
but to be honest I never tried to clone in such a big insert...

#7 scolix

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Posted 08 February 2012 - 07:43 AM

is it possible, you switched the ligation and control?

#8 kathyoc

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Posted 08 February 2012 - 07:50 AM

Nope, I definitely didn;t do that. I did 5 ligations and one control!!

#9 HOYAJM

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Posted 08 February 2012 - 08:23 AM

I would definately check 10 colonies just to make sure. I have had control and experimental plates have near the same number and I still got lucky and found a clone. But as bithorax mentioned I would increase the vector:insert ratio to even 1:8 or 1:10. This might be tricky since your vector and insert are about the same size.

#10 scolix

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Posted 08 February 2012 - 10:09 AM

Sometimes, the vector is over digested and cannot be ligated properly. If one of the enzymes has partially digested and other has over digested, there will be no positive colonies but negative colonies.




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