7 replies to this topic

[PJB]

I'm cloning an MCS oligo into a reporter plasmid, through which I introduced three new restriction sites into it. I'm now screening some colonies for these sites, but all the digests look the same: two RE's linearize the plasmid nicely, there's only one uncut band, but the third RE gives a linearized band and one that's 1kb HIGHER (bad MSpaint drawing attached). I have no idea what's causing this as there's no sign of contamination in any of the other lanes.

The only thing I can think of is that the extra band runs at twice the size of the supercoiled one, so it might be two plasmids ligated to eachother. However, this doesn't show in the uncut lane, and I don't think this can be caused by a restriction digest? RE3 is a blunt cutter, RE1 and 2 are sticky, if that matters.

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[pito]

What does no RE mean?
Is it a plasmid that you did not digest or a plasmid that does not have the restriction sites in it?

Also:

Quote

two RE's linearize the plasmid nicely, there's only one uncut band

, not sure what this means. There is only one uncut band? You are cutting... You mean: just 1 band, meaning the plasmid is just opened?

[PJB]

Yes, no RE means undigested plasmid. Because there is only one band in this lane, there is only one size of plasmid. The other three lanes are cut once, so they show linearized plasmid. However, cutting with RE3 gives a band that's higher than the total size of the plasmid...

At any rate, since our stock of RE3 is almost gone and it's clearly doing something strange, we've decided to proceed using the other two sites and ignore the weird cut RE3 produces, since the other three lanes are as expected.

[pito]

PJB, on 28 January 2013 - 08:10 AM, said:

Yes, no RE means undigested plasmid. Because there is only one band in this lane, there is only one size of plasmid. The other three lanes are cut once, so they show linearized plasmid. However, cutting with RE3 gives a band that's higher than the total size of the plasmid...

At any rate, since our stock of RE3 is almost gone and it's clearly doing something strange, we've decided to proceed using the other two sites and ignore the weird cut RE3 produces, since the other three lanes are as expected.

I am not really following you.

The other two lines are as expected? How can they be as expected?
They show a larger plasmid then the uncut plasmid...

Again: the plasmit with RE, it does mean it has the MCS , or do you mean an "unchanged" plasmid?

[Trof]

Uncut plasmid is probably mostly supercoiled, that's why it runs lower than single-cut. (but you should see other variants of uncut plasmid, can you post the real picture?)
The RE3 can also cut once and the higher band is nicked (resulting from an incomplete cutting), that can run higer than linear and supercoiled variant.





But that's just a theory.

I would cut longer with RE3, unless it's not suitable for it or is a bad cutter. Or just made it cut completely if possible.
Or cut plasmid with good cutting REs that cut more times, like 3 or 4, and check the band sizes, smaller lengths are easier to distinguish.

[PJB]

Trof, on 28 January 2013 - 09:11 AM, said:

The RE3 can also cut once and the higher band is nicked (resulting from an incomplete cutting), that can run higer than linear and supercoiled variant.

Thanks, this is probably what it is. In most preps, the top band is a lot brighter than the bottom one, to the point that the bottom one is almost absent. This site is one that I put in between the others to give the enzymes some space and to diagnose my ligation, I won't actually be using it for cloning. Maybe in the future I'll need to open up the plasmid again with blunt ends, so I'll be sure to keep it in mind!

[pito]

PJB, on 28 January 2013 - 09:20 AM, said:

Trof, on 28 January 2013 - 09:11 AM, said:

The RE3 can also cut once and the higher band is nicked (resulting from an incomplete cutting), that can run higer than linear and supercoiled variant.

Thanks, this is probably what it is. In most preps, the top band is a lot brighter than the bottom one, to the point that the bottom one is almost absent. This site is one that I put in between the others to give the enzymes some space and to diagnose my ligation, I won't actually be using it for cloning. Maybe in the future I'll need to open up the plasmid again with blunt ends, so I'll be sure to keep it in mind!

Altough its true what trof said, this should not be the case in this problem.

Secondly: you should run your gel long enough (which is pretty much always the case) that the problems with slower migration is not really a big problem. Even if this woud happen, you should see more then what you see, as trof also said.

See here: http://arbl.cvmbs.co...ls/agardna.html  or for a "real" image: http://www.nature.co...mbor346_f2.html

Are you sure you did everything right?


Is the image you made an image from 1 gel or you ran more then 1 and just made 1 image?

[PJB]

I ran multiple gels at about 4V/cm for 3 hours. All plasmids that did get cut by RE3 had the extra band, those that didn't had no 6kb band either. We've ruled out pretty much everything else, it has to be the enzyme itself. The nicked DNA migrating slower than linearized explains what we see perfectly.

I've attached a crappy cellphone picture of a printout of one of the gels. I hope you can make out the bands, DNA concentrations vary somewhat between preps and the bad camera on glossy paper doesn't help either. Every four lanes is a single prep. Obviously, the one on the left is from a bad colony, the other three are as I've drawn previously. In the middle one, there's some uncut plasmid left in two of the digests, but there aren't any other bands to be seen.

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