Neb Hifi Cloning issue - 15kb final plasmid
I'm attempting to replace a 1000bp sequence from my plasmid (9kb plasmid) with a different 7kb fragment (insert), to create a 15kb final plasmid. However, when I checked 6 colonies (so far) they are mostly linearised plasmid re-ligated, its not the template plasmid because the 1000bp has been removed correctly when I sequenced it. 1 of the colonies had the 7kb fragment inserted in it, but it also removed 5kb downstream of it, so 10kb in size instead of 15kb.
Do I just keep screening for more or I'm unlikely to find the correct construct? I double checked the primers for hifi and they are correct.
This is the reaction I did:
PCR linearised plasmid then gel purified, with good purity. 76 ng/ul concentration
PCR amplified 7kb insert with overhangs, also gel purified. 63ng/ul
10 µL NEBuilder reaction using NEBuilder® HiFi DNA Assembly Master Mix:
2:1 ratio of insert(7kb):vector(8kb)
15 min 50 degrees, then transformed 2ul into 50ul of neb10 beta, standard procedure, didn't try anything like 30 degrees instead of 37 degrees, not sure if that would help?
I can't avoid gel purification because of other bands appearing, but since I'm getting colonies without template DNA, I think its fine. would CIP help to prevent re-ligation of the linearised vector?