Ligation insert without PCR? - (Jan/11/2007 )
Can I Ligate fragment digested out of one plasmid construct directly to the second plasmid without PCR the insert fragment? Since the construct contains the correct fragment with two right restriction sites needed to ligate with the second plasmid. I am wondering just because in my memory PCR insert is always necessary before it is ligated into plasmid. I guess it is only because restriction sites need to be created at both ends of the insert by PCR. But if the fragment has already have those sites in the first construct, can I directly cut it out and ligate to the second plasmid?
yup yes you can cut out an fragment and provide the ends of said insert are compatible, ligate the insert into a second plasmid.
You do need to gel purify your insert first, to remove the orginal vector.
Absolutely, this is one of the best things in cloning. u dont have to PCR every time. Just follow perneseblue's advice.
Verify that the sites are not present inside the insert.
It's even best not to PCR it if you can cut it out one plasmid to ligate into another. PCR can lead to unwanted mutations which is not the case with restriction-purification-ligation.
I think I have made it. Thank you all!
except if you burn your gel under the UV light... that produces T dimers (I know one case of UV mutations after gel purifucation) but if you work properly, don't worry about it!!!
if there is some tag before MCS you want to insert into, be careful of the frame. otherwise no problem.