4 replies to this topic

[serundipity2009]

Hello,

I amplifed my 700bp fragment with PCR and now I need to ligate a 700bp fragment to a 6kb vector using xhoI and NdeI sites. Would 90 min at RT or 90 min at 4C work?
Also, do i need to gel purify my pcr reaction before ligation? Will it work if i just use my pcr reaction directly or do pcr purify?

Thanks alot

[noelmathur]

Unless I am in hurry, I ligate at 15C overnight. Otherwise 2 hours at RT should work. Just make sure that your ligase buffer isn't thawed and frozen lot of time (as you would have less ATP and ligation would fail.)

[fishdoc]

I incubate T4 DNA ligase (NEB) reactions at room temp anywhere from from 2 hours to overnight, and it works fine.

[amelia417]

You should purify your PCR product before use to be safe. Prior to ligation, I have run the DNA for ligation on a gel, cut it from the gel, and purified it from the gel, and then did the ligation.

[phage434]

Just making sure: you must clean up your pcr reaction prior to the digestion you are doing on the pcr fragment. If you do not clean up the pcr reaction, the restriction enzymes will cut and the site will fill in with the pcr enzyme and dNTPs left over from the pcr. The restriction digest can often be heat killed rather than purified, and I would recommend that unless the enzymes cannot be heat killed.