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Ligation with small insert and large vector

ligation small insert large vector

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6 replies to this topic

#1 luckyjade

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Posted 14 May 2013 - 08:19 PM

I am having trouble with ligation with a small insert (800bp) and a large vector (13,000bp). I am currently using three fold molar excess insert. So 20ng insert to 100ng vector. When I plate it for transformation, no colonies grow at all.
I have also tried 20ng insert to 70ng vector. I do not think the problem is with the vector or the insert. Once again, no colonies grow.
Does anyone have any ideas on other combinations?

Edited by luckyjade, 14 May 2013 - 11:11 PM.


#2 christy

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Posted 14 May 2013 - 08:26 PM

http://www.insilico..../Lig_Input.html

In this ligation calculator you can try 1:5 or 1:10 ratio of Vector:Insert. The ratio of vector and insert depends on the size.

#3 luckyjade

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Posted 14 May 2013 - 08:38 PM

http://www.insilico..../Lig_Input.html

In this ligation calculator you can try 1:5 or 1:10 ratio of Vector:Insert. The ratio of vector and insert depends on the size.


How do you know to try 1:5 or 1:10? How does the size alter the ratio and how is this particular ratio determined?? Thank you for your help by the way!

Edited by luckyjade, 14 May 2013 - 08:40 PM.


#4 phage434

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Posted 15 May 2013 - 07:43 AM

This is (in my experience) very rarely the problem, although it gives people who can't get ligation to work something to talk about. Sort of like Tm calculation of primers for PCR. Tell me about your controls -- transformation efficiency, especially. What evidence do you have that your insert and vector are not the problem?

#5 luckyjade

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Posted 15 May 2013 - 07:43 PM

I plated 5 ng of DNA (a previous DNA that was digested and then ligated) using competent cells from the same batch and lots of colonies grew. Does ng of DNA amount matter? I thought it was more ng-->more colonies

#6 phage434

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Posted 16 May 2013 - 05:12 AM

5 ng of DNA should produce at least a million colonies if you have highly competent cells. Check your transformation efficiency with 10 pg of plasmid DNA, and calculate the efficiency. You should get 10**8 to 10**9 colonies per microgram of DNA.

#7 christy

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Posted 17 May 2013 - 07:09 PM

Yes right. The competant cell efficiency is very important. And one more thing is the host organism.





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