I didn't get my clone (pcDNA3.1+insert+GFP). I cloned GFP successfully but my promoter cloning is very difficult . I replaced CMV promoter with my promoter (2 different promoter (1).930 BP and (2).2085 BP) . Overall size 6.33 KB and 7.48 KB (2 Construct) . My restriction and elution, I got a clear band of CMV nearly 757 BP.
1. Ligation buffer has precipitated means, what can i do?
2. Can I use ATP (cofactor) with NEB or Fermentas buffer for ligation reaction?
3. What amount of ATP is required?
4. Can I use PCR machine (Eppendorf) for overnight ligation (16`C)?
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