Problem on restriction digest - Inquire (Nov/01/2006 )
Hi guys, i cloned a 300 bp insert into a 9 kb vector after double digesting it with EcoR1 and BamH1. After ligation and electroporation, I was able to get 2 transformants and did a miniprep afterwards. After which i digested it again with EcoR1 and BamH1 and another sample with EcoR1 only to confirm the success of my experiment.
The gel showed the expected size for the uncut plasmid, but those that are cut with EcoR1/BamH1 and EcoR1 showed no bands. I did the digestion for 3 hours at 37C using NEB No. 2 buffer.
When i checked it via PCR using primers for the vector, i get the expected size again for my insert which is a little over than its original size.
Now i dont understand, y i cant digest it again..
any comments?thanks
The gel showed the expected size for the uncut plasmid, but those that are cut with EcoR1/BamH1 and EcoR1 showed no bands. I did the digestion for 3 hours at 37C using NEB No. 2 buffer.
When i checked it via PCR using primers for the vector, i get the expected size again for my insert which is a little over than its original size.
Now i dont understand, y i cant digest it again..
any comments?thanks
Are you sure that the problem isn't the fact that you could loose your band? How long did you run the gel? Is the EtBr still present where you should see the 300bp band? Try to run the gel for not too long and see if you have the expected band; let it run longer and look at it again. If you don't see the band, try to stain with EtBr and see if you can detect the band
Hi guys, i cloned a 300 bp insert into a 9 kb vector after double digesting it with EcoR1 and BamH1. After ligation and electroporation, I was able to get 2 transformants and did a miniprep afterwards. After which i digested it again with EcoR1 and BamH1 and another sample with EcoR1 only to confirm the success of my experiment.
The gel showed the expected size for the uncut plasmid, but those that are cut with EcoR1/BamH1 and EcoR1 showed no bands. I did the digestion for 3 hours at 37C using NEB No. 2 buffer.
When i checked it via PCR using primers for the vector, i get the expected size again for my insert which is a little over than its original size.
Now i dont understand, y i cant digest it again..
any comments?thanks
Are you sure that the problem isn't the fact that you could loose your band? How long did you run the gel? Is the EtBr still present where you should see the 300bp band? Try to run the gel for not too long and see if you have the expected band; let it run longer and look at it again. If you don't see the band, try to stain with EtBr and see if you can detect the band
Also, a 300bp band will only be 1/30th as bright as a 9 kb band, under ideal conditions. You may need to overload that sample.
Also, do you really mean no bands at all in the digested samples? Or, is there a 9kb band but no 0.3 kb band?
what gel density did you use? How much of the miniprep DNA did you cut?
As already mentioned 300bp fragment doesn't flores as brightly as a larger DNA fragment. It can easily be missed and if you are running a 1% agarose gel, almost certainly lost.
For a situation like this I would do PCR to check for the inserts presence. And I would do a digest using a restriction enzyme that cuts only within the insert. That way you can tell if the insert is in the vector or not. If the DNA cuts the insert is present. If the plasmid DNA doesn't cut then the insert is absent
Thanks for the reply guys, i really appreciate it. i ran the samples on a 1% agarose gel for 30 minutes 100V...and yes there were no bands at all. not even the vector. ill try to attch later the pics of my gel..
thanks
As already mentioned 300bp fragment doesn't flores as brightly as a larger DNA fragment. It can easily be missed and if you are running a 1% agarose gel, almost certainly lost.
For a situation like this I would do PCR to check for the inserts presence. And I would do a digest using a restriction enzyme that cuts only within the insert. That way you can tell if the insert is in the vector or not. If the DNA cuts the insert is present. If the plasmid DNA doesn't cut then the insert is absent
hi, i digested 5 ul of the miniprep with EcoR1 and BamH1, and 2 ul of the miniprep using EcoR1 only...
The PCR showed the insert using primers for the vector sequence, but in the digestion it didnt.
what gel density did you use? How much of the miniprep DNA did you cut?
As already mentioned 300bp fragment doesn't flores as brightly as a larger DNA fragment. It can easily be missed and if you are running a 1% agarose gel, almost certainly lost.
For a situation like this I would do PCR to check for the inserts presence. And I would do a digest using a restriction enzyme that cuts only within the insert. That way you can tell if the insert is in the vector or not. If the DNA cuts the insert is present. If the plasmid DNA doesn't cut then the insert is absent
hi, i digested 5 ul of the miniprep with EcoR1 and BamH1, and 2 ul of the miniprep using EcoR1 only...
The PCR showed the insert using primers for the vector sequence, but in the digestion it didnt.
Just to be clear, did you run some of your uncut miniprep as well?
A pic would really help.
One thought I have had is that maybe you didn't purify your clone (restreak it before inoculating broth), and are using amp - and that during growth some satellites (amp sensitive, no plasmid) took over the culture. Amp is degraded by a few amp resistant cells, and amp sensitive cells can then grow, if they're present.
If that was the case, then you'd have a teeny itty bit of plasmid (enough to PCR from but not really enough to visualize on a gel.)
You could see if your plasmid has a different resistance marker (kan is good) and grow up in that instead. It's less likely to be broken down by the resistant cells, and so there is less of a problem with satellites. Of course, it is always a good idea to purify your clones ---- restreaking them on LB amp before picking to broth.....
what gel density did you use? How much of the miniprep DNA did you cut?
As already mentioned 300bp fragment doesn't flores as brightly as a larger DNA fragment. It can easily be missed and if you are running a 1% agarose gel, almost certainly lost.
For a situation like this I would do PCR to check for the inserts presence. And I would do a digest using a restriction enzyme that cuts only within the insert. That way you can tell if the insert is in the vector or not. If the DNA cuts the insert is present. If the plasmid DNA doesn't cut then the insert is absent
hi, i digested 5 ul of the miniprep with EcoR1 and BamH1, and 2 ul of the miniprep using EcoR1 only...
The PCR showed the insert using primers for the vector sequence, but in the digestion it didnt.
Just to be clear, did you run some of your uncut miniprep as well?
A pic would really help.
One thought I have had is that maybe you didn't purify your clone (restreak it before inoculating broth), and are using amp - and that during growth some satellites (amp sensitive, no plasmid) took over the culture. Amp is degraded by a few amp resistant cells, and amp sensitive cells can then grow, if they're present.
If that was the case, then you'd have a teeny itty bit of plasmid (enough to PCR from but not really enough to visualize on a gel.)
You could see if your plasmid has a different resistance marker (kan is good) and grow up in that instead. It's less likely to be broken down by the resistant cells, and so there is less of a problem with satellites. Of course, it is always a good idea to purify your clones ---- restreaking them on LB amp before picking to broth.....
hi thanks for the reply. actually i restreaked the clone before using the broth, and i run the uncut plasmid from the miniprep as can be seen in the pictures attached. thank u so much for the comments
using a 1kb ladder
first image shows 3 lanes. 1st lane is the insert, the 2nd and 3rd are from the plasmid using vector sequence primers
2nd image shows 6 lanes. lanes 1 and 4 are uncut plasmids from the miniprep, while the 2,3,5,6 lanes are cut with EcoR1 and BamH1 and Eco R1 only.
Is lane four on gel one, the negative PCR control?
It looks like you have plasmid (lanes 1 and 4, gel 2) and that those plasmids have the right size insert (lanes 2 and 3, gel 1) , but that when you do a restriction digest you 'lose' the plasmid. Sound right?
I've never heard of that happening, and I am wondering if there is some sort of DNAse contamination or something. Also, I assume you didn't try to precipitate the DNA after restriction digest or anything like that, did you? I assume you loaded the digest directly onto the gel, yes?
It looks like your cloning worked, but that your restriction digest was screwy.
You could try a couple things. You could try PCR'ing from your miniprep using the original PCR primers (sort of one more confirmation that you really have the right insert), but you really need to get your plasmid DNA digest figured out. Maybe you could do a larger prep?
Is the plasmid a low copy number plasmid? 9 kb is big, and the cells might not like making too much of it. I used to work with a 13 kb plasmid, and I think its copy number was around 10 per cell. The yields were always very low. You could try some other miniprep procedures, or a larger scale prep. Next time you do the digest, use fresh reagents (to avoid any posasible DNAse contamination, though I've never heard of that happening) and include a different plasmid prep as a positive control for digestion/ getting the DNA at the end of the digestion.
Sorry I can't really help out too much --- It's definitely a puzzle.
It looks like you have plasmid (lanes 1 and 4, gel 2) and that those plasmids have the right size insert (lanes 2 and 3, gel 1) , but that when you do a restriction digest you 'lose' the plasmid. Sound right?
I've never heard of that happening, and I am wondering if there is some sort of DNAse contamination or something. Also, I assume you didn't try to precipitate the DNA after restriction digest or anything like that, did you? I assume you loaded the digest directly onto the gel, yes?
It looks like your cloning worked, but that your restriction digest was screwy.
You could try a couple things. You could try PCR'ing from your miniprep using the original PCR primers (sort of one more confirmation that you really have the right insert), but you really need to get your plasmid DNA digest figured out. Maybe you could do a larger prep?
Is the plasmid a low copy number plasmid? 9 kb is big, and the cells might not like making too much of it. I used to work with a 13 kb plasmid, and I think its copy number was around 10 per cell. The yields were always very low. You could try some other miniprep procedures, or a larger scale prep. Next time you do the digest, use fresh reagents (to avoid any posasible DNAse contamination, though I've never heard of that happening) and include a different plasmid prep as a positive control for digestion/ getting the DNA at the end of the digestion.
Sorry I can't really help out too much --- It's definitely a puzzle.
hi, nope i didnt precipitate the DNA after digestion. i loaded it to the gel immediately after digestion.
ill take note of your helpful advice. i might as well start PCR using the original primers and see where it will take me.. thank u so much patty