Frustrated with Plasmid Preps - need help! - (Jan/29/2009 )

scolix on Feb 1 2009, 12:04 PM said:

Interesting. I ordered a pCAG-DsRed from AddGene. Hopefully, the CAG promoter will give much higher efficiency. What vector did you end up using?

I had my student run a gel on one of the Clontech plasmids (CMV-AcGFP) that is not working and an older plasmid (CMV-Rac1-GFP) that was working several months ago, but now is not working. It doesn't look like a great gel, but I think you can see that the linearized DNA migrates to approximately the correct size (5 kb). However, the uncut DNA appears to be primarily in two bands, which I assume the supercoiled form migrates lower (faster). Unfortunately, it is the other band in both instances that appears brighter. Does this indicate there is indeed a problem with our prep?

Can anyone help me with this? It seems the uncut DNA is running slower than the linearized DNA - that's not good, right? What kinds of things could cause this outcome.

brightfield on Feb 7 2009, 01:40 PM said:

Often linearized plasmid runs between supercoiled and relaxed, but for large plasmids, linearised can run faster. It may varied whether the plasmid is associated with or without salts, mono and divalent ions. I don't think you can tell if there is a problem with your plasmids with a single cut. You need to sequence it, but I double it's the expression problem you are having.

To your question on other post -"if prechilling the cell culture is needed before centrifuge," the answer is "No."

AquaPlasmid on Feb 7 2009, 04:58 PM said:

These two plasmids are only ~5 kb.