are you facing any problem? You may get my contact no from Aman sinha of your Dept. for detailed discussion.
Yes sir i am facing difficulty in cloning of my gene. I will contact dr aman may be if he have your contact number.
Hi I used pET 101 TOPO vector from invitrogen. You can as well detail the problem you are encountering
bro, i have problem with ligation of my gene. Transformation and ligation of control product as well as puc dna given with kit is going well. But when my topo ligation reaction take place it shows no colony on plate. I have incubate at 22 degree celsius from 30 min to overnight but still there is no growth of colony with my insert. I have even checked the primer twice but they are also accurately designed with CACC at ends. What can be the best method for cloning of such large fragment with this kit?
Hi Kanisht,
From your information its clear your components are working fine. You said your primers were accurately designed, did you say you have CACC at the ends, the CACC should only be at the forward primer not both ends. The length of your amplicon should not be a problem as I have successfully cloned a 2kb amplicon before moreover the supplier once told me that I can clone larger fragment using any of the kit vectors. I hope you are using a high fidelity Polymerase due to the large amplicon. I will advise you sequence your PCR product to be sure that mutation is not introduce during amplification which could affect your cloning. I wish you success
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