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Generating stable cell line with linear DNA

stable linear ligation

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3 replies to this topic

#1 miketibs

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Posted 05 March 2012 - 08:55 AM

Hello,

I'm planning on generating a stable cell line using linearized plasmid DNA. Is there a risk of having my DNA re-ligate? If so should I treat my DNA with alkaline phosphatase after digestion? Please let me know how I would do this as I am new to all of this.

Thanks,

#2 miketibs

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Posted 05 March 2012 - 09:14 AM

Also, is there a difference between blunt and sticky end digestion?

#3 bob1

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Posted 05 March 2012 - 11:51 AM

There is a small risk of re-ligation, though you are more likely to get concatamerization, and the amount of re-ligated DNA without adding a ligase will be very very low, so I wouldn't worry about it.

It shouldn't matter whether you use blunt or sticky, but blunt is less likely to re-ligate.

#4 miketibs

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Posted 06 March 2012 - 07:03 AM

Thanks bob1. Fingers crossed.





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