Hello Gaurav,

Is your pET28a vector having His tag after the multiple cloning site? If so it will be in frame with your MCS so that, it too gets translated along with the gene you cloned. There are two things here

1. For the purpose of cloning in pET28a vector, if you have PCR amplified your gene initially using forward and reverse primers having the sites you mentioned, check whether you designed the reverse primer where you have removed the stop codon of your gene and added extra bases between your last codon of your gene and SacI site if needed so that it is reading in frame with the His tag.

2. Also is your forward primer designed such that after the XbaI site whether you have added extra bases if needed so that it will read in frame with the vector you are using and reading frame is not disturbed for the vector codon and also your gene.

For the vectors which have tag after MCS we have to be a bit careful so that your tag also gets read. Write back if you have any queries.

Good luck with your cloning

You do realize that by using the XbaI site you've eliminated all translational initiation signals from the vector, right?

As Arjan advised, you can try any combination of filling-in (partial or not) or resecting with T4 DNA pol; not necessarily with the Sac I site (which generates a 3'OH-protruding end) but with the downstream sites at the MCS as well. In my opinion, however, you might solve your problem faster and with less hassle by simply redesigning the reverse PCR primer and repeating the cloning experiment.

Thank you  every body  for your valuable suggestions. I have redesigned the reverse primer with Hind III  to avoid the further complexities. I have got the clone with right frame.