clarification - plasmid and gel - (Nov/24/2005 )
Can anyone tell me once and for all what is the difference when running an uncut plasmid between the super coiled, linear and other that I don't know...
and what are the "rules" for the different agarose percentage that should be used for separation.
thanks a lot
Supercoiled: the normal state of plasmids inside coli cells. Carefully done preps contain mostly supercoiled plasmid. These run faster on a gel than the cut linear plasmid fragment.
Nicked plasmid: results from damage to the supercoiled DNA resulting in loss of supercoiling. Often this is nicking a single strand. Runs more slowly than a linear DNA of the same length. Produced by ligating DNA into a circular plasmid when the vector has been treated with CIP or SAP.
Relaxed or covalently closed DNA: Can be produced by using topoisomerase I enzyme acting on supercoiled plasmid DNA. This is the form produced on ligating DNA into a circular plasmid. Runs at a similar speed as nicked plasmid.
Linear DNA: result of double strand nick or restriction enzyme cutting of supercoiled or relaxed plasmid.
Denatured plasmid: strands are not paired over the full length. Runs more slowly than linear or relaxed plasmid.
We almost always use a 1% agarose gel, with the NEB 2-log ladder. For short fragments (200 -1000) use 1.5%; for long fragments > 4 KB use 0.6 - 0.8%. For very small fragments use 2-4% Nusieve 1:3 or Metaphor agarose, or switch to PAGE.
I pretty much agree with phage434...
There are three major bands produced by separating uncut plasmid DNA. In order, from slowest running to fastest running, they are relaxed or nicked circular (supercoling not yet accomplished [relaxed] or removed [nicked, a single stranded break]), linear (double stranded break), and supercoiled. There are also sometimes other forms visible: dimeric forms of the three species listed above, and sometimes, if you leave your preparation in the alkaline lysis buffer too long, there are denatured forms visible as well.
The relaxed form can be minimized by not harvesting your cells too early, the nicked form can be reduced by handling your preparations gently (this will reduce the linear form as well) and completing the plasmid recovery prep without delay. Some forms can be reduced by choosing another host (using endA- strains like DH5alpha versus endA+ strains like HB101 or BL21, for example), and formation of plasmid dimers, while not usually a big problem, is strain and vector dependent.
Maximizing the supercoiled form is always helped by working quickly.
Maybe applicably to this thread (job-oriented learning...):
case 1): after a mini-prep two bands can be seen on the gel: one at ~10 kb, another one at
> 23 kb. My vector has a size of roughly 10 kb, so what could the bigger band be?
irreversibly denatured plasmid? But does it really run that slowly? And why does the smaller
one run at 10 kb although it's still circular?
case 2): yet another plasmid isolation, same vector (10 kb). There's one band at ~ 6 kb. After
linearizing it runs at 10 kb. Does supercoiling make such a big difference?
Would be nice if someone could help me to figure out, what happened...
For question 1:
If you're running uncut plasmid, and comparing it to a linear standard, there's no way to estimate size (except for the linearized plasmid portion). So, even if your upper band is migrating through the gel the same distance as your 23 kb linear standard, this does not mean it's 23 kb. Most likely, it's nicked or relaxed circular plasmid.
For question 2:
The smaller plasmid band is not 6 kb of DNA, but migrates through the gel to the same extent as your 6 kb linear standard (using the same logic as above). It is most likely undigested supercoiled plasmid DNA.
Yes, I was aware of that, only found it surprising that there's such a big difference in the migration behaviour between the relaxed and the supercoiled plasmid, that's all.
And also: why does a circular vector run the same distance as a linear molecule of the same size (what it apparently did)?
Thanks for clarification so far.
Let me add one (last?) question to this thread:
I recently have done site directed mutagenesis on a 3.5 kbp plasmid, using Novagens Quikchange Kit. After DpnI-restriction with 10u for 1h at 37°C (getting rid of the template) i´ve checked the success of the PCR on an agarose gel. What I see is an intensive band at ~3.5 kbp and one weaker band at around double the size.
What could this be ? Can these be two conformations of the same plasmid as well ? I´m just wondering, since Quikchange is supposed to produce nicked plasmids, so I´m not too sure whether the intensive band really is my desired product...
Well, I have similar problem about distinguish plasmids. I isolated enterococci DNA and ran gel. I got this picture and I thought all bands showed here are plasmid except one for chromosomal DNA. My supervisor told me I cannot say all count as plasmids because without RE. i don't know what was wrong. Could you anyone explain for me?
Could you post your picture as a .jpg, .gif, or .png file? The image in the Word document requires a QuickTime decompressor or some other such Macintosh thingy.
But, without even seeing the picture, I'm leaning towards your supervisor's opinion. If it's a plasmid, you should be able to isolate it without also recovering chromosomal DNA. Cutting it with a restriction enzyme is no big deal, either.
I could likely go from an overnight culture to a gel picture of a digested sample of recovered plasmid in what -- four hours?
Thanks for your answer HomeBrew.
Attached is ge pic with .JPG.
Lanes are 1: 1Kb ladder, 2-6: Enterococcus faecalis.
Please help me again, whether the bands are representing plasmids or not and to answer to advisor bravely.