5 replies to this topic

[cellgene]

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?

[Vini]

cellgene, on Mar 18 2010, 07:40 PM, said:

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

[Curtis]

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.

[perneseblue]

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.

[cellgene]

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

[cellgene]

thank you again

my cloning worked