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keeping restriction digested fragments at -20?


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5 replies to this topic

#1 cellgene

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Posted 18 March 2010 - 05:10 AM

I have a bunch of digested PCR products(with sticky ends) and I kept them at -20 for 4 days now.
Can they be used for ligation?

#2 Vini

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Posted 18 March 2010 - 07:38 AM

I have a bunch of digested PCR products(with sticky ends) and I kept them at -20 for 4 days now.
Can they be used for ligation?



yeah sure, you can use them :D

#3 Curtis

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Posted 18 March 2010 - 08:49 AM

you can, but better avoid freezing before ligation. It is recommended to ligate right after digestion. That's how we do it in our lab in order to keep the efficiency high.

#4 perneseblue

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Posted 18 March 2010 - 04:05 PM

how big are your fragments?

if they are just 5-10kb you can keep them for months. I have ligated DNA fragments that were prepared a year ago with no noticeable difference. It is a different story if your are ligating fragments in the +50kb range. It is better to keep those fragments at 4C, they don't seem to like repeated freeze thawing cycles.
May your PCR products be long, your protocols short and your boss on holiday

#5 cellgene

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Posted 19 March 2010 - 02:32 AM

thank you all!

i ligated them before freezing but there were some problems so i repeat now. they are slightly bigger than 1kb. i am doing now so i will inform you about the result:)

i didn't know it is better to keep at 4degrees; from now on i keep in mind :)

#6 cellgene

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Posted 27 March 2010 - 05:22 AM

thank you again

my cloning worked :D




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