Hi An easy fix is to PCR amplify your low copy number plasmid (modern polymerases have no issues amplifying up to 20 kB if you have a largeish vector) and then sequence the product. Alternatively you can amplify just a portion of interest, of course (you said sequence the plasmid so I went with your exact request). The spectionmycin trick has already been proposed - that also works well :)
Now, you also mentioned that you would like to quantify the plasmid - for that a simple qPCR (or even a carefully tuned regular PCR) will do the trick, or if you want to go seriously old school you can quantify it by dilution, transformation, and colony counting against a known standard plasmid. Happy new year Artem On Wed, Dec 30, 2020, 4:35 AM Anamika Singh <anamika.ii...@gmail.com> wrote: > Hi All, > > I have two constructs having different ori, p15ori and M13 ori, different > promoters araBAD promoter and LacI, and different antibiotic resistance > chloramphenicol and Ampicillin respectively. > I am managed to get the transformants and getting the expected result > after blunt digestion with the EcoRV enzyme. Since both the plasmids have > the site for EcoRV. But the p15 ori has a low copy number that's why I am > seeing the very faint band as compared to other plasmid in the sample. > > So I would like to know is there any way that I can quantify the low copy > number plasmid. > Because I am not able to sequence it with the specific primers it could be > due to its low concentration. > > Please advise. > > Thank you. > -- > Dr. Anamika Singh > Post-Doctoral Fellow > Silberman Institute of Life Sciences > Hebrew University of Jerusalem, Israel > No: 054-294-8036 > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
