We are trying to transform pBC vector in DH5alpha . We follow normal transformation procedure which involves heat shock at 42 degrees for 45 seconds . But we are not getting any colonies. As pBC has bigger size do we need to change the protocol? And also we have smaller quantity of pBC vector what are the methods available to check if we want to confirm plasmid quantity and characteristics
Since you have plasmid, I would run both uncut plasmid on a gel (100 or 200 ng) to make sure that it's intact plus do a digest with a RE that cuts your pBC into 4 or so easily discernible bands on a gel to confirm that it is what you think it is.
Then, make sure you have a positive control plasmid for ensuring that the competent cells are competent. If you have a plasmid with the same selectable marker as your pBC, you can use it to test that your selection is working and not overly stringent (such as if the plates were made with 10X antibiotic by mistake). Plus, there are Amp and Chlor versions of the pBC vectors and there may have been a mislabeling of the tube you have such that your selection isn't suitable for the marker in that plasmid. The digest above, if the RE is chosen carefully, may reveal which version of the plasmid is in your tube prior to transformation.
If the controls are working but you're still not getting colonies from an intact and correct-looking-by-digest pBC, then there may be something in the plasmid prep that's inhibiting the transformation. You can repurify it on a column and transform with different amounts. Hopefully, you're only transforming 20 - 100 ul of cells with 0.1 - 1 ng of plasmid and then outgrowing the cells for 1 hour at 37 while shaking before plating.
Good luck.
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