How to determine the size of the insert in the vector.

[kirupha]

Hi all,

I am working with PCC1BAC vector which contains my insert. I have sent the BAC DNA to sequencing to determine the size of the insert. I have got back the sequencing results and using serial cloner software I'm trying to determine the insert size. Can anyone help me with this.

Thanks,
Kirupha

[Olaf Blue]

The pCC1BAC vectors are all 8,128 bases no matter the linearizing nuclease:

http://www.epibio.com/tech-support/dna-sequences/pcc1bac_hind

Otherwise digest the clone with the enzyme NotI as it cuts ~300 bases either side of the insertion site. Will generate a ~7.5 kb vector band and at least one insert band depending on the insert sequence and NotI recognition sites. Run on a 0.7-1.0% agarose gel, stain, photograph, measure migration using a ruler and suitable sizing program.