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Ligation issues


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#1 amisra2

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Posted 02 January 2011 - 06:00 PM

So i have been having issues for the past couples of months to make my constructs.

I have a 15kb Viral vector (its a coronavirus inserted into pBR322) and I have a PCR product of 3.3kb. Both of which I cut with EcoRV and PacI, Gel purify both bands (I end up getting a pretty good amount for both which I have quantified by nanodrop and by running on a gel) and set up ligation reaction and transform. For some reason I keep getting about 10 to 20 colonies with a 1500bp band and not my desired band. I don't know what is going wrong?

I have tried the ligation reaction with different ratios of vector and isert and also at room temperature and 16degrees overnight....or just for an hour. For some of the colonies I can see a higher band at about 19kb which is what I want but it is a pretty faint band and the 1.5kb band is brighter. Please help!

#2 pDNA

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Posted 03 January 2011 - 01:41 AM

viral DNA is always prone to contain repeat sequences like inverted or direct repeats that tend to be unstable due to recombination in E. coli ...maybe your problems are due to DNA sequence instabilities?
What E. coli strain are you using for cloning? You can try to use Stbl or Sure cells (they can be purchased from Invitrogen i think). Check your insert for inverted or direct repeats or homologies to the vector backbone.
To increase your ligation efficiency you can also try using a ligase that is optimized for large constructs, e.g. Takara DNA Ligation Kit Long

Regards,
p

So i have been having issues for the past couples of months to make my constructs.

I have a 15kb Viral vector (its a coronavirus inserted into pBR322) and I have a PCR product of 3.3kb. Both of which I cut with EcoRV and PacI, Gel purify both bands (I end up getting a pretty good amount for both which I have quantified by nanodrop and by running on a gel) and set up ligation reaction and transform. For some reason I keep getting about 10 to 20 colonies with a 1500bp band and not my desired band. I don't know what is going wrong?

I have tried the ligation reaction with different ratios of vector and isert and also at room temperature and 16degrees overnight....or just for an hour. For some of the colonies I can see a higher band at about 19kb which is what I want but it is a pretty faint band and the 1.5kb band is brighter. Please help!






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