sequantial digest - (Jun/18/2014 )

Yes. Read the notes on T4 DNA Ligase (M0202) at NEB. Ligase is active in any of their buffer, when you supplement the buffer with 1 mM ATP.

Ok thanks a lot.

Than I'll try your tactic!

Thanks a lot.

phage434 on Tue Jun 24 18:26:16 2014 said:

Yes. Read the notes on T4 DNA Ligase (M0202) at NEB. Ligase is active in any of their buffer, when you supplement the buffer with 1 mM ATP.

Oh, one more thing: you mentioned something about cycling between 37°C and 16°C?

I know that for the T4 ligase it should be done at 16°C (but thats colder than our room temperature and thus it will be hard to archieve this, we dont really have a cooling system for this)

So I'll just use room temperature.

Is there any specific way to do this switching? Start with digesting for half an hour, than half an hour of ligating... and so on? or ?

phage434 on Tue Jun 24 18:26:16 2014 said:

Yes. Read the notes on T4 DNA Ligase (M0202) at NEB. Ligase is active in any of their buffer, when you supplement the buffer with 1 mM ATP.

We run digestions and ligations on a PCR cycler, which can easily snd quickly switch from 16 to 37 and back.

Ah yes!

Thats a good idea.

One more question: if you do the digest and ligase reaction together, dont you worry that the first cut (with the first RE) will be ligated again? It can be religated if the second RE does not remove the part I want to kick out of the plasmid.

phage434 on Tue Jun 24 21:53:03 2014 said:

We run digestions and ligations on a PCR cycler, which can easily snd quickly switch from 16 to 37 and back.

Yes, that could happen. We use it for simultaneous cutting with all of the enzymes and ligation with DNA which will destroy the restriction site(s).