Hy everyone, I have been working hard to clone an Arabidospis promoter in the pDONR p4-p1,r Entry Vector, but it didn't work until now !!!
When checking my primers, I obseved that Attb4 primer lacks a G nucleotide at the 5-primer (only 3 Gs instead of 4 Gs as recomended in the Gateway Manual). My questions: Is there a problem with this? BP Clonase reaction could affected if you have this situation in your PCR product?
I need some help, I dont know what I do. I obtained some positives clones when plating in LB Kanamycin 50 ug/uL, but the clones were negative for insert by PCR or Digestion.
Thak you so much !!
Hey Helder, I think the deletion of one base pair (G) of ur promoter leads to frame shift so that the promoter will not work
I hope my answer helps u.
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