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blunt end ligation problem


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#1 mahdijp

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Posted 09 June 2009 - 07:04 PM

Hi,
I am working on blunt end ligation of 3.5 kb DNA fragment using Mighty cloning kit. I gel purified my fragment and checked the purified fragment by running another electrophoresis of 2 ul of my fragment. Surprisingly, I had only few blue colonies. Sometimes also white colonies appeared but did not have my fragment. The technique that I am using here is applied to a 2.5 kb fragment and every thing worked out.
Any ideas why I having this problem which bugs my for 2 monthes.

#2 perneseblue

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Posted 12 June 2009 - 04:00 AM

Could you tell us exactly what you have done. Give every detail. Right now, there isn't enough information to tell you what might have gone wrong.

The best I can say is that something not quite right is happening to your insert (Perhaps it was the processing, it could well be there is insufficient insert being use- I need more details to tell). There really isn't much difference cloning a 2.5kb fragment and a 3.5kb fragment.
May your PCR products be long, your protocols short and your boss on holiday

#3 mahdijp

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Posted 17 June 2009 - 03:10 AM

Thank you very much of your reply. I do not know what kind of details do you need. I would really appreciate your help by giving me some hints and questions that might be behind all that mess.
Initially I am trying to join 3 fragments by overlapping PCR. With the first 2 fragments (totally 2 kb) everything worked out. But when I am trying to join the 3rd fragment I got no clone. I have one notice here that after joining the first 2 fragment with the 3rd one I got a band not that much intensive. However, I check my fragment every time after gel extraction by running 2ul of gel-purified amplicon.




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