I am trying to clone 3 bits generated by pcr, and all three are nearly 0.8kb, one of which is chloramphenicol gene. I need to clone the other 2 bits flanking the antibiotic gene at either ends.
I have tried,
1. ligating all three bits at once,.... too many bands ( expected 2.4 kb)
2. ligated Ist bit with antibiotic gene, and same way with other one ( expected 1.6kb each). Although I got 1.6 kb, but when I checked by pcr with fwd primer of Ist bit and Rv primer of antibiotic gene, I did not get any 1.6 kb product. So it looked like similar bits were ligating.
I have also changed the REN sites, but no use.
Could someone help me please. I've been trying this fro past 3 months, but no result.
thanks.
shilpa
