Weird migration of double digested plasmid - (Feb/03/2011 )

Hi Everyone,
After a long pause I am back to cloning. I am facing a strange issue. I am using a plasmid of size 5151 bp for double digestion with NdeI and XbaI. The sites are seperated by 33bp. Immediately after double digestion I treated the plasmid with antarticphosphatase(NEB) for 30 mins and then phenol extracted before I ran it on the gel to purify the linearised form. The problem I see in the form of two bands, none of them corresponding to original size of the vector. I used uncut plasmid in a parallel lane as a control to eliminate any undigested product in my lane, but none of the two bands correspond to the uncut plasmid (isn't that somewhat weird?). The slow migrating band is appx. 8 kb sized and faster one appx. 4 kb sized. I gel extracted both of them (to save some time). But, now I am puzzled, which one would be the right band that I must choose to use for ligation. Please, shed some light on this that would help me to understand the problem and avoid any mistakes in the future. Particularly if anyone knows how I ended up with these strange bands.
Thanks a lot for everyone's effort.

I'd guess that your plasmid is not what you think it is. You could clarify things by cutting with each enzyme separately, which would verify that each cuts, and also tell you the length of the parent plasmid. This is a much better way of establishing plasmid length than running uncut plasmid, which can run at many different speeds, depending on supercoiling and nicking. Are you sure the plasmid has not already had an insert? You might be seeing plasmid backbone and insert as two bands. Where did the plasmid come from?

phage434 on Fri Feb 4 01:09:28 2011 said:

I see, you got a point. I haven't run a routine analysis of parent plasmid because I found them in our stock and never used after purchase.
quote "Are you sure the plasmid has not already had an insert? You might be seeing plasmid backbone and insert as two bands." in that case also it has to give at least one band at the size that I am expecting because as I mentioned above the empty plasmid is around 5 kb and it has only one of each NdeI and XbaI sites.

Even today I ran another digestion analysis for shorter time(1hr), I got nothing improved but too many bands of unexpected size. Even one close to the range 6 kb. In this case somehow I want to belive that when the plasmid is linearized due to its huge size and overloaded well (appx. 90ug of starting material)it migrates bit higher when compared to the marker, is that possible?

Anyway, I will run a check for plasmid authenticity first. Thanks for your suggestion.

I have done several restriction digestions to confirm the parent plasmid even after single and double digestions, the size remained more or less the same wired way it was.

I have attached the digestion gel and the vector map of the plasmid.

The legend for DNA digestion gel

lane 1: uncut plasmid
lane 2: NEB smart ladder 10 Kb
lane 3: single digestion with EcoRI
lane 4: single digestion with NdeI
lane 5: single digestion with XbaI
lane 6: free
lane 7: Double digestion with EcoRI and NdeI
lane 8: Double digestion with EcoRI and XbaI

Even if I ignore the results from lane 8 as improper digestion, the results are still not what we expect. After the confusing digestion result even I did sequencing starting from the promotor to check any possible inserts. I have got nothing more than the expected GST tag in that. Now it looks more wired. Anyone got any idea!

Your input is very much appreciated! Thanks a lot

Explain exactly how you are preparing your DNA and exactly how you set up your digestions. What are your buffers, amounts of DNA, enzyme, buffer, water, do you use BSA. How do you purify your DNA? Are you using the NEB -HF enzymes?

EcoRI often has star activity, and can cut at sites other than the ones you expect. These are the bands I see in your gel (assuming the ladder is really NEB 2-log ladder):
1 - supercoiled, ignore
2 - marker
3 - 6.2Kb + 3.3 Kb (partial digestion of this band, probably star activity, possibly two overlapping bands)
4 - 6.5 Kb
5 - 6.5 Kb
6 -
7 - 2.9 Kb + 2.6 Kb + 1.3 Kb (difficult to interpret, perhaps more complete digestion of a star site + the insert you expect)
8 - EcoRI enzyme probably absent from this digestion

It seems very likely your plasmid has 6.5 - 7 Kb of DNA, not the 5100 you expect. Somewhere in that additional DNA is either a real EcoRI site or one with strong star activity with EcoRI under your cutting conditions.

It seems unlikely your original NdeI + XbaI digest really showed an 8 Kb band. Do you have a gel for that? It would be good to repeat the NdeI + XbaI and EcoRI + XbaI digestions. Putting a second marker lane near those would also be helpful.