clone selection

[wahbar]

Hi everyone,

I'm getting colonies for my ligation. but when it comes to selection, im getting
different results. I'm using colony PCR to screen the clones n also doing RE digestion. colony pcr gives me the correct band size for my insert which is around 3kb. however RE digestion of those same clones which i got a band for does not show the same band.
does this mean that the RE sites r not there? but if is not then how did the ligation take place? both the vector n insert has been cut with both NdeI and BamHI.

[mchlbrmn]

Hi everyone,

I'm getting colonies for my ligation. but when it comes to selection, im getting
different results. I'm using colony PCR to screen the clones n also doing RE digestion. colony pcr gives me the correct band size for my insert which is around 3kb. however RE digestion of those same clones which i got a band for does not show the same band.
does this mean that the RE sites r not there? but if is not then how did the ligation take place? both the vector n insert has been cut with both NdeI and BamHI.

If the insert cloned in with only 1 enzyme, because one enzyme wasn't working well, (or there was a second site for 1 enzyme?) you would linearize the DNA with the RE cut. That would make 1 large band the total size of the vector+insert. Supercoil circular usually gives a smaller size band, plus some fainter bands usually for a fraction of the plasmids being nicked relaxed circles, or linear.

Sometimes the end(s) of the DNA insert and vector can be damaged, and the DNA might clone in blunt, with the overhangs knocked off, or in some other odd way. This can destroy the RE site(s). In this case if you use REs that are both inside the vector some distance from the cloning site, you can now see the insert size difference.

Is it possible your colonies still have enough plasmid DNA from the ligation around them to PCR? (I don't much experience with looking by PCR). Or, are you sure it's the correct band and not a secondary false band? Did you have a no plasmid control colony?

[relaxin]

As mchlbrmn mentioned, the RE sites were lost because the vector sequences flanking the cloning site were degraded by nonspecific nucleases during vector preparation. To avoid this problem, you may use primers based on the vector sequences flanking the cloning site in the initial colony sscreening. Then confirm the orientation of the insert using one vector-specific primer and one insert-specific primer.

[wahbar]

If the insert cloned in with only 1 enzyme, because one enzyme wasn't working well, (or there was a second site for 1 enzyme?) you would linearize the DNA with the RE cut. That would make 1 large band the total size of the vector+insert. Supercoil circular usually gives a smaller size band, plus some fainter bands usually for a fraction of the plasmids being nicked relaxed circles, or linear.

Sometimes the end(s) of the DNA insert and vector can be damaged, and the DNA might clone in blunt, with the overhangs knocked off, or in some other odd way. This can destroy the RE site(s). In this case if you use REs that are both inside the vector some distance from the cloning site, you can now see the insert size difference.

Is it possible your colonies still have enough plasmid DNA from the ligation around them to PCR? (I don't much experience with looking by PCR). Or, are you sure it's the correct band and not a secondary false band? Did you have a no plasmid control colony?

hmm the band i got for the RE is bigger than the vector size yet smaller than the vector+insert. and also some supercoiled plasmid. ok so u mean i shud try some other RE some distance away from the cloning site to check whether i get a band of correct size? for the colony PCR, i had used half a colony for PCR and half for patching onto a plate. so i would grow cultures from there n extract plasmid.

[wahbar]

As mchlbrmn mentioned, the RE sites were lost because the vector sequences flanking the cloning site were degraded by nonspecific nucleases during vector preparation. To avoid this problem, you may use primers based on the vector sequences flanking the cloning site in the initial colony sscreening. Then confirm the orientation of the insert using one vector-specific primer and one insert-specific primer.

sorry i'm not too sure what u mean by vector preparation and when the RE sites were degraded. i need to do some protein expression work after this so i have to check whether the insert is inframe. so if the insert is there but the RE sites r not that means it won't be inframe? so how should i avoid this degradation? thanks.

[WeirdOmen]

hmm the band i got for the RE is bigger than the vector size yet smaller than the vector+insert. and also some supercoiled plasmid.

This means that you're using only one enzyme in your check, right? (you got only one band...) Are you absolutely sure that the sizes don't match? If it was me, i'd double-digest it, to be sure.

The "eating away" of the ends is something i've heard of in this forum but that I've never encountered personally, so i suppose it's not that common. Personally I'd be more inclined towards mchlbrmn's second advice,

Is it possible your colonies still have enough plasmid DNA from the ligation around them to PCR? (I don't much experience with looking by PCR). Or, are you sure it's the correct band and not a secondary false band? Did you have a no plasmid control colony?

this is something i've seen sometimes. You see a faint positive on PCR and think that perhaps the colony was small, but when you try growing and digesting, your insert isn't there - the PCR had amplified the ligation DNA, and not plasmid inside your bacteria.

[relaxin]

sorry i'm not too sure what u mean by vector preparation and when the RE sites were degraded. i need to do some protein expression work after this so i have to check whether the insert is inframe. so if the insert is there but the RE sites r not that means it won't be inframe? so how should i avoid this degradation? thanks.

When you digest your vector with RE, the contaminating nucleases in the RE may chew off some sequence from the cut ends. Some how your insert can still be ligated to these degraded vector molecules. If you check your colonies using insert-specific primers, you find positives with the insert. You should recheck those positives with primers based on the sequence of the vector flanking the cloning site. Only those with intact vector sequence will give you a positive band.

If you cannot cut the insert with RE, the cloning sites are gone, your expressed protein will not be in-frame. Chances are the promoter may also be degraded, and you will have no expression at all.

Therefore, you should check the clones with two sets of primers as described above, before you attempt any expression experiments.

[mchlbrmn]

Well, I guess my first paragraph on rereading isn't very comprehensible, but that option wasn't very likely anyway.

We seem to agree that because of the possibility the cloning sites are damaged, you should use PCR primers or RE sites inside the vector, and look for the correct PCR product or a correct shift in the size of the restriction enz. fragment. I suppose this confirms that you have the insert cloned in the vector. It would be good to run a little uncut DNA as a control, ( I didn't understand what you meant by, "and also some supercoiled plasmid. "), and it would be ideal to confirm that the REs are working by cutting maybe some empty vector. Then you'll also confirm where vector and supercoil are running. Oh, I'm assuming WeirdOmen misunderstood you and you were using 2 REs. What enzymes were they,by the way, and what buffer did you use?

An off the wall idea. Suppose the insert wouldn't clone at all, and 2 vectors ligated to each other to recircularize as a dimer. The supercoil form of that might run smaller then vector + insert but larger then linear vector.

If you confirm inserts, or even if you don't, to be sure what you have you could send some to sequence with primers set in 100 bp, or so, into the vector. If you have time, that's the most definitive. Possibly, if the cloning sites are gone the clone is no good. There's some chance it's only lost some junk sequence around the intact ORF (reading frame) and it is OK.

Oh, and in my hands plasmid purified from bacteria off a plate came out fairly crappy. It might not be very good for some REs, or sequencing. I suppose if you/your lab do this routinely you might know if it's OK.

I'd also double check that the REs don't cut internally inside the insert (or vector).

If the insert cloned in with only 1 enzyme, because one enzyme wasn't working well, (or there was a second site for 1 enzyme?) you would linearize the DNA with the RE cut. That would make 1 large band the total size of the vector+insert. Supercoil circular usually gives a smaller size band, plus some fainter bands usually for a fraction of the plasmids being nicked relaxed circles, or linear.

Sometimes the end(s) of the DNA insert and vector can be damaged, and the DNA might clone in blunt, with the overhangs knocked off, or in some other odd way. This can destroy the RE site(s). In this case if you use REs that are both inside the vector some distance from the cloning site, you can now see the insert size difference.

Is it possible your colonies still have enough plasmid DNA from the ligation around them to PCR? (I don't much experience with looking by PCR). Or, are you sure it's the correct band and not a secondary false band? Did you have a no plasmid control colony?

hmm the band i got for the RE is bigger than the vector size yet smaller than the vector+insert. and also some supercoiled plasmid. ok so u mean i shud try some other RE some distance away from the cloning site to check whether i get a band of correct size? for the colony PCR, i had used half a colony for PCR and half for patching onto a plate. so i would grow cultures from there n extract plasmid.

[wahbar]

thanks for all the replies so far. it has given me sth to think about.

previously have tried sequencing to check whether the RE sites were intact but so far the sequencing has failed but some reason so still troubleshooting on that.

Relaxin, u said to try using a vector primer which flanks the cloning site. does that mean there is still hope of finding some colonies which might be postitive? or could it be that all of the clones have the RE sites being degraded? n if that is the case how should i repeat my experiments to prevent this from taking place. The enzymes i used are from Fermentas, NdeI and BamHI and the vector i'm using is pET vector. I'm cloning the insert in the MCS so shouldn't have any problem of RE sites at other places in vector.

Tomorrow i will try to cut with other RE to confirm for the insert.

[relaxin]

Relaxin, u said to try using a vector primer which flanks the cloning site. does that mean there is still hope of finding some colonies which might be postitive? or could it be that all of the clones have the RE sites being degraded?

There are always some good ones. If you still have colonies left on the plate, by all means check for presence of insert using primers bases on the vector sequence. If the size of the band is as expected, purify the poistive colony by streaking, and then check for orientation of the insert, using sense primer based on vector and antisense primer based on insert.