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Ligation problem


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#1 Marbaum

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Posted 03 October 2013 - 01:14 PM

Hi All,

 

I am trying to insert a fragment in the .THT vector, which was cut with two RE : NdeI and BamHI. Then double cut vector is gel purified and  "ligated" with insert using T4 ligase. After transforming ligated vector, it was digested again to see if the insert will be present on the gel. And the result for this was negative. So we tried to CIP treat vector before ligation and that did not work either. 

 

Then we decided to vary ratio of DNA:insert (1:1, 1:3, 1:57, 1:15) and none of those worked. So I will be very grateful if you make any suggestion how to make this ligation to work.

 

Thanks in advance.

 

 



#2 jerryshelly1

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Posted 03 October 2013 - 01:42 PM

What are your vector and insert sizes? Also, what are your incubation conditions? Did you use a control to verify your ligase is working?



#3 Marbaum

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Posted 03 October 2013 - 01:50 PM

My vector is 5300 bp and insert is around 400 bp. Incubation conditions: digestion was carried at 37C for 2 hours and ligation was done at 16C overnight. Right after ligation I did transformation and initially thought that the only thing that should be there is my vector with insert. But number of colonies was way too high, so we did different ratio of DNA:insert. Number of colonies there was in almost linear dependence to the increasing amount of insert. So I thought that was a good sign. Although, after miniprepping several colonies and digesting them we could see only double cut vector on the gel and no insert. 

 

I would appreciate if you can tell me what control I can use for ligation. 

 

Thanks.



#4 phage434

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Posted 03 October 2013 - 02:02 PM

How was your insert prepared?



#5 Marbaum

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Posted 03 October 2013 - 02:04 PM

It was genomic PCR reaction that was double cut with same RE: NdeI and BamHI, gel purified after. The gel results showed the length that was right.



#6 jerryshelly1

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Posted 03 October 2013 - 02:04 PM

Sometimes if an restriction enzyme has been sitting unused for sometime, I will test each one by taking a known concentration of a plasmid and i will double check the efficiency of each enzyme (Add 5U of enzyme to whatever concentration of DNA and us HindIII ladder to see ng of linearized DNA). From here, digest my insert, CIP it and then ligate it for 16C for 2 hrs, 4C overnight, and 16C for 2 hrs. Depending on the quantity of my ligation mixture, I will either run it on a gel and gel extract my ligated product or just directly transfect it.

 

Good controls:

1) Plasmid without insert to verify activity of ligase.

2) Plasmid without insert and without ligase to check the activity of your CIP.

 

I usually use these controls to verify all of my enzymes are functional.

 

A good thing to check is your primer design.  Are you using an insert that was produced via PCR? Did you design the primers to produce an overhang that will provide maximum efficiency of your enzyme (reference - https://www.neb.com/...f-dna-fragments).

 

 

Does this help?



#7 Marbaum

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Posted 03 October 2013 - 02:12 PM

Thanks.

 

Now by looking at what controls you meant I can tell you that I did both of what you mentioned, transformed them. So the results were following:

 

1) plasmid without insert + ligase produced a lot of colonies ( I mean really a lot)

2) I did plasmid without ligase and without CIP + insert and it produced some colonies after the transformation.

 

I actually did not do the plasmid without insert and without ligase but with CIP treatment. And this sound like a good idea, I will definitely try it. So the result of this one should be no colonies on the plate because there is no phosphate that can be used to make bond again? Is that right?



#8 jerryshelly1

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Posted 03 October 2013 - 02:15 PM

You are correct. Everything sounds correct. Your PCR product was developed to have a sufficient overhang for digestion?

 

After you digested your plasmid, did you isolate your linearized product via gel purification?



#9 Marbaum

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Posted 03 October 2013 - 02:20 PM

I am not sure about the primers because I did not prepare them.

 

But my digested plasmid was gel purified. And it run a little higher than uncut plasmid, and this fact makes sense. Supercoiled DNA runs lower than the one that was digested and now linearized.



#10 phage434

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Posted 03 October 2013 - 03:59 PM

Check your primers for having sufficient 5' overhangs outside the restriction enzyme cut site.

 

You can verify cutting of your pcr product. Purify your PCR product, then cut with one of your two enzymes. Heat kill the enzyme (if possible, else purify it). Ligate just this cut PCR product. You should see a double length fragment. Do this with the other enzyme as well. Now you know that both of your PCR ends are correctly digested.



#11 Marbaum

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Posted 04 October 2013 - 04:03 AM

Just checked my primers and they have 3 nucleotides overhang, that should be enough for RE that I am using.  






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