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PCR Profile for ligation


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#1 samita

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Posted 28 October 2013 - 06:48 AM

Does someone have a PCR profile for ligation reaction???

 

 



#2 bob1

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Posted 28 October 2013 - 11:51 AM

You mean a thermal cycler profile?  PCR is polymerase chain reaction, and quite different to ligation.

 

The profile on a thermal cycler would be dependent on how you want to do your reaction (room temp?, 16 deg? 4 deg?) and how long you want to do your reaction (generally shorter for higher temps).  I generally use 16 deg with a hold until I tell it to stop (usually overnight).



#3 phage434

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Posted 28 October 2013 - 03:15 PM

I usually ligate on the bench for 15-30 minutes. Ligations with overhangs almost always work well, without optimization, if the DNA fragments are correct and at the appropriate concentration.



#4 samita

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Posted 29 October 2013 - 02:17 PM

Few years ago I used PCR for ligation step. Now I lost the profile and never did cloning for a long time so forget every thing about cloning now.






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