I've been having problems with cloning since 4 months now, the vector seems to self-ligate with itself for some reason without taking in the insert tried different res sites and different vectors but the problem is still not solved. I've given here the steps that I followed.
· Insert (5.8kb) and Vector (3.5kb, pSL119)
· Plasmid isolation (using Qiagen mini prep)
· Phusion PCR amplification of Insert from plasmid using non phosphorylated primers to add res sites to ends (7bp overhangs).
· Gel extraction of the fragment (Using Thermo scientific gel ext kit)
· Restriction of the Fragment using Nco1 and BamH1 (Fast Digest, Thermo Scientific ) (should produce phosphorylated cohesive ends) also tried with (Sal1 and BamH1)
· Reaction Purification (using Qiagen Reaction purification Kit)
· Vector Dephosporylated using Fast AP
· (Thinking of using kinase on the insert now, haven’t done it yet)
· Overnight Ligation using T4 DNA Ligase (1:5, Vector:Insert) (also tried 1:2, 1:3, 3:1, 2:1)
· Reaction 1: Vector=20ng; Insert: 100ng
Reaction 2: Vector=100ng; Insert=500ng
· Produces several colonies and all the colonies have self-ligated vector and no insert (colony PCR analysis and analysis by restriction digestion)
Getting really frustrated with this hope you guys can help.