Sequencing

Sequence is cloned to p Bluescript vector and transformed to E.coli. Selected cloned aquired ampicillin resistant , but plamid is not visible in agarose gel. I wish to know what could be the reason? How to increase copies of plasmid?

I need to know a little more detail about what you have actually done before I can help trouble shoot this. pBluescript is a relatively high copy number plasmid, so it seems unlikely that copy number is an issue - unless for some reason, your insert happens to be toxic to e.coli...

Did you perform the correct controls for your e.Coli transformation? It is possible you are looking at a contamination and not bacteria transformed by your plasmid of interest. (A "bugs only" control helps with this.) Are you sure you added ampicillin?

How are you checking for the presence of the plasmid? If you are trying to perform PCR directly on bacterial colonies, you will need to troubleshoot your PCR reaction, or consider growing mini-preps to check for plasmid.

If you have done mini-preps, trouble shoot the mini-prep protocol by including a control culture from something you know works. Check for the presence of DNA by spectrophotometry, then be sure you are using enough DNA so as to be visible in an agarose gel (at least 200 ng.)

Mini-preps have a reputation for poor quality DNA, so keep in mind that it may hamper your ability to perform PCR or a digest. If you are having trouble with mini-preps, consider going straight to Maxi preps. I think they are more reliable (especially when it comes to Qiagen.)

Write again when you figure it out and let us know!