5 replies to this topic

[The Question]

Hello everyone

I was wondering about which plasmid DNA to use for standard curve generation. Circular plasmid DNA, linear plasmid DAN (after digestion with one restriction enzyme) or PCR product of the circular DNA?

Any experiences, suggestions please!

Thanks!

[Eve]

What do you want to measure and how are you planning on measuring it?

If it is gel-based, I would use linear since circular dna can supercoil and give two bands.

[stylothecancer]

Hi The Question,

I presume you are talking about real time pcr here when you actually post your question here, hope i am right.

Linearized plasmid.

This is because if the plasmid is not linearized, it would affect the PCR efficiency even though your primers and probes have been properly designed.

[MariC]

Hi!
after linearizing plasmid, do I have to purify it or can I directly do the dilutions and use them as standards for my experiments?
Thank you for any advice!
MC

stylothecancer, on Feb 4 2010, 02:27 AM, said:

Hi The Question,

I presume you are talking about real time pcr here when you actually post your question here, hope i am right.

Linearized plasmid.

This is because if the plasmid is not linearized, it would affect the PCR efficiency even though your primers and probes have been properly designed.

[vladooo]

You probably need to know plasmid copy numbers so you have to purify it first, measure on nanodrop and then caluclate how many copies per ng you have and then create serial dilutions accordingly.

[tea-test]

I never linearized plasmids for standard curves. The plasmid only serves in the first cycle as template, than you are anyway amplifying from linear PCR product.