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Vector-insert ratio


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#1 Raygoza

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Posted 26 July 2014 - 08:28 AM

I know that the recommendation to select the molar ratio between vector and insert in a ligation reaction is to run a gradient. However isn't there a relation (function, formula) that would always work between the relative sizes? the problem is that i have a 5.6kb vector and i want to insert a 2.6kb insert, but i'm not having any luck in doing so.

 

thanks



#2 phage434

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Posted 26 July 2014 - 10:12 AM

It is unlikely that an different ratio will solve your problem. Almost certainly, something else is the problem. Tell us (in detail) what you are trying to do, and exactly how you are trying to do it.



#3 Raygoza

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Posted 26 July 2014 - 11:12 AM

Ok. So first i'm amplifying a region of the genome which is 2.6kb, adding the necessary restriction sites in the primers and enough bases after the sites. so far I'm stuck with this size because this wasn't designed by me. Then the vector is 5.6kb in length and i digest both the insert and vector with KpnI-HF and SacI-HF from NEB (again stuck with this for the moment). I can confirm the enzymes both work in individual restriction reactions. I perform the double digest for an hour, i have tried overnight and everything is fine by gel. First I was getting too much background re-ligation, so i moved into using alkaline phosphatase on the vector only, however then i stopped getting any colonies. All the cleaning i've been doing it using spin columns, but someone told me to do it with gel extraction but i'm just afraid to lose everything but maybe someone can tell me if this is more effective. So i moved into antarctic phosphatase and according to the gel nothing was being ligated. For ligation I use T4 ligase for 1 hour at room temperature. Note that i never use phosphatase on the insert. I've tried 3:1, 5:1 and 7:1 insert:vector ratios. with no success so far. I hope someone can help me get out of this puddle. 


Edited by Raygoza, 26 July 2014 - 11:13 AM.


#4 phage434

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Posted 26 July 2014 - 12:14 PM

How much DNA are you adding to your ligation mix? Too much DNA can be a problem. Try in the vicinity of 20 ng of vector and 1-3 x as many molar molecules of insert. More is not better.

How are you transforming? Again, too much ligation mix can inhibit transformation. 1-2 ul per 50 ul of competent cells. More is not better.

I would not plan on seeing ligation products on a gel. Directly transform, and don't worry about what the gel says.

If you are using antarctic phosphatase, use as little as possible, and limit the time -- pay attention to the units (one of the few times you need to).

 

You could test the ligation of your insert. After PCR, cut with KpnI (only) and purify. Ligate (only the purified insert), and look for a double length product. Do this again for SacI. If you fail to see a double length product, then there is a problem in the digestion of your PCR product. You already know your vector is being cut from looking at the insert released on double digestion.



#5 Raygoza

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Posted 26 July 2014 - 03:28 PM

I've tried with  lot at the beginning but then i went down to 10ng, maybe that's the reason.



#6 seanspotatobusiness

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Posted 07 August 2014 - 03:03 AM

Phage is way more qualified to help than I am but could ligation proceed better at 16 C?



#7 phage434

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Posted 07 August 2014 - 06:51 AM

You can certainly increase the efficiency of ligation by going to lower temperatures, but in routine cloning, ligation is sufficiently fast and efficient that 15 minutes at room temperature works (with cohesive ends).






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