10 replies to this topic

[biobio]

Hello all!


I am trying to cut my vector with XhoI and SpeI. I setup a digestion reaction like this:

3.3 ul template (1ug)
2 ul 10x NEB Buffer 4
2 ul 10x BSA (I prepared this by diluting 100x stock in water, not 1X buffer)
1ul XhoI
1ul SpeI
11.7 ul H20
Incubation 37 C for 1h, inactivation 65 C for 20 min.

Then I run on a gel the cut and uncut vector.

In each lane, I got two top bands that were quite faint, definitely not representative of 1ug. I also got some fat bright bands but these were very small size, they run past last band of ladder.

So my questions are:

Why am I seeing such a small amount of vector and backbone? I re-speced the template and im definitely loading 1 ug.

I dont see an insert or even linearized vector released. But I get with such low amounts maybe it's not visible? I think the bands suggest some cutting takes place, cause cut and uncut top bands are slightly different size.

What are the fat bottom bands? Is it possible it's all star activity and the whole template gets chopped up in small fragments. I think it's unlikely with those amounts of enzyme. Also, this band is present in the uncut vector!

I am beginning to suspect there is something fundamentally wrong with my template. Any suggestions? :/

Edited by biobio, 19 September 2009 - 03:25 PM.

[Warren]

biobio, on Sep 19 2009, 07:24 PM, said:

Hello all!


I am trying to cut my vector with XhoI and SpeI. I setup a digestion reaction like this:

3.3 ul template (1ug)
2 ul 10x NEB Buffer 4
2 ul 10x BSA (I prepared this by diluting 100x stock in water, not 1X buffer)
1ul XhoI
1ul SpeI
11.7 ul H20
Incubation 37 C for 1h, inactivation 65 C for 20 min.

Then I run on a gel the cut and uncut vector.

In each lane, I got two top bands that were quite faint, definitely not representative of 1ug. I also got some fat bright bands but these were very small size, they run past last band of ladder.

So my questions are:

Why am I seeing such a small amount of vector and backbone? I re-speced the template and im definitely loading 1 ug.

I dont see an insert or even linearized vector released. But I get with such low amounts maybe it's not visible? I think the bands suggest some cutting takes place, cause cut and uncut top bands are slightly different size.

What are the fat bottom bands? Is it possible it's all star activity and the whole template gets chopped up in small fragments. I think it's unlikely with those amounts of enzyme. Also, this band is present in the uncut vector!

I am beginning to suspect there is something fundamentally wrong with my template. Any suggestions? :/

those fat bottom band(s) that run faster than the ladder sound like RNA to me.  Is this miniprepped DNA?  If your "spec" comes from using some type of absorbance measurement and you have a boatload of RNA in there, your probably not getting 1 ug of your DNA but about that amount of total nucleic acid, of which only a small amount is your DNA.  Warren..

[biobio]

Warren, on Sep 19 2009, 05:02 PM, said:

those fat bottom band(s) that run faster than the ladder sound like RNA to me.  Is this miniprepped DNA?  If your "spec" comes from using some type of absorbance measurement and you have a boatload of RNA in there, your probably not getting 1 ug of your DNA but about that amount of total nucleic acid, of which only a small amount is your DNA.  Warren..

Hi Warren, thanks for reply.

I think you may be on to something.. I didn't prepare the plasmid myself, but it's was almost certainly isolated with a Qiagen Maxi or Mega Kit. I use a nanodrop to measure nucleic acid concentration. I am not 100% sure, but I think the 260/280 ratio was indicative of DNA.. (1.7-1.9)

Could it be something else, like chromosomal DNA from the bacteria? But I think this would show a band high on the gel, no?

In any case, how would I overcome this - I really don't want to prepare the template again. Should I just try adding more of it (for example 10x more, judging by the uncut band intensity) and treat it as digestion of 1 ug (so add only 1 ul of enzyme)?

Edited by biobio, 19 September 2009 - 04:24 PM.

[Warren]

biobio, on Sep 19 2009, 08:12 PM, said:

Warren, on Sep 19 2009, 05:02 PM, said:

those fat bottom band(s) that run faster than the ladder sound like RNA to me.  Is this miniprepped DNA?  If your "spec" comes from using some type of absorbance measurement and you have a boatload of RNA in there, your probably not getting 1 ug of your DNA but about that amount of total nucleic acid, of which only a small amount is your DNA.  Warren..

Hi Warren, thanks for reply.

I think you may be on to something.. I didn't prepare the plasmid myself, but it's was almost certainly isolated with a Qiagen Maxi or Mega Kit. I use a nanodrop to measure nucleic acid concentration. I am not 100% sure, but I think the 260/280 ratio was indicative of DNA.. (1.7-1.9)

Could it be something else, like chromosomal DNA from the bacteria? But I think this would show a band high on the gel, no?

Chromosomal DNA from bacteria runs very high and often doesn't migrate much out of the well using normal gels.  Its either RNA or your DNA is degraded.  Very simple way to test -- you should have some RNase A around (it comes with alot of these kits, just make sure it is DNase free if it wasn't from one of these kits)....add a tiny bit to a sample of your DNA, and run it again on a gel (incubate maybe five minutes)....if those fat bands are RNA they will have disappeared or be significantly reduced.  Warren..

[biobio]

Warren, on Sep 19 2009, 05:23 PM, said:

biobio, on Sep 19 2009, 08:12 PM, said:

Warren, on Sep 19 2009, 05:02 PM, said:

those fat bottom band(s) that run faster than the ladder sound like RNA to me.  Is this miniprepped DNA?  If your "spec" comes from using some type of absorbance measurement and you have a boatload of RNA in there, your probably not getting 1 ug of your DNA but about that amount of total nucleic acid, of which only a small amount is your DNA.  Warren..

Hi Warren, thanks for reply.

I think you may be on to something.. I didn't prepare the plasmid myself, but it's was almost certainly isolated with a Qiagen Maxi or Mega Kit. I use a nanodrop to measure nucleic acid concentration. I am not 100% sure, but I think the 260/280 ratio was indicative of DNA.. (1.7-1.9)

Could it be something else, like chromosomal DNA from the bacteria? But I think this would show a band high on the gel, no?

Chromosomal DNA from bacteria runs very high and often doesn't migrate much out of the well using normal gels.  Its either RNA or your DNA is degraded.  Very simple way to test -- you should have some RNase A around (it comes with alot of these kits, just make sure it is DNase free if it wasn't from one of these kits)....add a tiny bit to a sample of your DNA, and run it again on a gel (incubate maybe five minutes)....if those fat bands are RNA they will have disappeared or be significantly reduced.  Warren..

Yeap, we have some RNase around - will try that, good suggestion!

Let's assume I do have RNA contamination or DNA degradation, how would I overcome this - I really don't want to prepare the template again. Should I just try adding more of it (for example 10x more, judging by the uncut band intensity) and treat it as digestion of 1 ug (so add only 1 ul of enzyme)?

[Warren]

biobio, on Sep 19 2009, 08:26 PM, said:

Warren, on Sep 19 2009, 05:23 PM, said:

biobio, on Sep 19 2009, 08:12 PM, said:

Warren, on Sep 19 2009, 05:02 PM, said:

those fat bottom band(s) that run faster than the ladder sound like RNA to me.  Is this miniprepped DNA?  If your "spec" comes from using some type of absorbance measurement and you have a boatload of RNA in there, your probably not getting 1 ug of your DNA but about that amount of total nucleic acid, of which only a small amount is your DNA.  Warren..

Hi Warren, thanks for reply.

I think you may be on to something.. I didn't prepare the plasmid myself, but it's was almost certainly isolated with a Qiagen Maxi or Mega Kit. I use a nanodrop to measure nucleic acid concentration. I am not 100% sure, but I think the 260/280 ratio was indicative of DNA.. (1.7-1.9)

Could it be something else, like chromosomal DNA from the bacteria? But I think this would show a band high on the gel, no?

Chromosomal DNA from bacteria runs very high and often doesn't migrate much out of the well using normal gels.  Its either RNA or your DNA is degraded.  Very simple way to test -- you should have some RNase A around (it comes with alot of these kits, just make sure it is DNase free if it wasn't from one of these kits)....add a tiny bit to a sample of your DNA, and run it again on a gel (incubate maybe five minutes)....if those fat bands are RNA they will have disappeared or be significantly reduced.  Warren..

Yeap, we have some RNase around - will try that, good suggestion!

Let's assume I do have RNA contamination or DNA degradation, how would I overcome this - I really don't want to prepare the template again. Should I just try adding more of it (for example 10x more, judging by the uncut band intensity) and treat it as digestion of 1 ug (so add only 1 ul of enzyme)?

If it is RNA, I would try as you suggest (the RNA shouldn't cause problems) but if your template is degraded, I would just prepare new template.  You may be able to transform cells with the prep if it is not too far gone, or maybe someone has glycerol stocks of bacteria with the plasmid and you can just grow some.  Preparing new template is easy (except the wait) and you can use miniprep DNA for most things you need to do, so no need to do a large scale purification.  Bad plasmid will cause you so many headaches trying to do cloning, that you will waste alot of time and figure out later you need to purify some new stuff anyway.  The good news is, that sounds like RNA bands to me!  Warren..

[biobio]

Warren, on Sep 19 2009, 05:39 PM, said:

biobio, on Sep 19 2009, 08:26 PM, said:

Warren, on Sep 19 2009, 05:23 PM, said:

biobio, on Sep 19 2009, 08:12 PM, said:

Warren, on Sep 19 2009, 05:02 PM, said:

those fat bottom band(s) that run faster than the ladder sound like RNA to me.  Is this miniprepped DNA?  If your "spec" comes from using some type of absorbance measurement and you have a boatload of RNA in there, your probably not getting 1 ug of your DNA but about that amount of total nucleic acid, of which only a small amount is your DNA.  Warren..

Hi Warren, thanks for reply.

I think you may be on to something.. I didn't prepare the plasmid myself, but it's was almost certainly isolated with a Qiagen Maxi or Mega Kit. I use a nanodrop to measure nucleic acid concentration. I am not 100% sure, but I think the 260/280 ratio was indicative of DNA.. (1.7-1.9)

Could it be something else, like chromosomal DNA from the bacteria? But I think this would show a band high on the gel, no?

Chromosomal DNA from bacteria runs very high and often doesn't migrate much out of the well using normal gels.  Its either RNA or your DNA is degraded.  Very simple way to test -- you should have some RNase A around (it comes with alot of these kits, just make sure it is DNase free if it wasn't from one of these kits)....add a tiny bit to a sample of your DNA, and run it again on a gel (incubate maybe five minutes)....if those fat bands are RNA they will have disappeared or be significantly reduced.  Warren..

Yeap, we have some RNase around - will try that, good suggestion!

Let's assume I do have RNA contamination or DNA degradation, how would I overcome this - I really don't want to prepare the template again. Should I just try adding more of it (for example 10x more, judging by the uncut band intensity) and treat it as digestion of 1 ug (so add only 1 ul of enzyme)?

If it is RNA, I would try as you suggest (the RNA shouldn't cause problems) but if your template is degraded, I would just prepare new template.  You may be able to transform cells with the prep if it is not too far gone, or maybe someone has glycerol stocks of bacteria with the plasmid and you can just grow some.  Preparing new template is easy (except the wait) and you can use miniprep DNA for most things you need to do, so no need to do a large scale purification.  Bad plasmid will cause you so many headaches trying to do cloning, that you will waste alot of time and figure out later you need to purify some new stuff anyway.  The good news is, that sounds like RNA bands to me!  Warren..

I see. Will try the RNase step and take it from there. I still find it slightly weird that I would have so much RNA there.. Is it a common problem with Qiagen preps?

Another related (pretty basic) question, as I know that XhoI doesnt cut well if site is methylated. If I wanted to get rid of methylation by growing plasmid again in Dam- bacteria, could I use the (methylated) plasmid I already have? Or do I have to go one step back to a non-methylated stock and use that for transformation?

Sorry to keep pestering you with questions! Thanks again for all your help..

[Warren]

biobio, on Sep 19 2009, 08:49 PM, said:

Warren, on Sep 19 2009, 05:39 PM, said:

biobio, on Sep 19 2009, 08:26 PM, said:

Warren, on Sep 19 2009, 05:23 PM, said:

biobio, on Sep 19 2009, 08:12 PM, said:

Warren, on Sep 19 2009, 05:02 PM, said:

those fat bottom band(s) that run faster than the ladder sound like RNA to me.  Is this miniprepped DNA?  If your "spec" comes from using some type of absorbance measurement and you have a boatload of RNA in there, your probably not getting 1 ug of your DNA but about that amount of total nucleic acid, of which only a small amount is your DNA.  Warren..

Hi Warren, thanks for reply.

I think you may be on to something.. I didn't prepare the plasmid myself, but it's was almost certainly isolated with a Qiagen Maxi or Mega Kit. I use a nanodrop to measure nucleic acid concentration. I am not 100% sure, but I think the 260/280 ratio was indicative of DNA.. (1.7-1.9)

Could it be something else, like chromosomal DNA from the bacteria? But I think this would show a band high on the gel, no?

Chromosomal DNA from bacteria runs very high and often doesn't migrate much out of the well using normal gels.  Its either RNA or your DNA is degraded.  Very simple way to test -- you should have some RNase A around (it comes with alot of these kits, just make sure it is DNase free if it wasn't from one of these kits)....add a tiny bit to a sample of your DNA, and run it again on a gel (incubate maybe five minutes)....if those fat bands are RNA they will have disappeared or be significantly reduced.  Warren..

Yeap, we have some RNase around - will try that, good suggestion!

Let's assume I do have RNA contamination or DNA degradation, how would I overcome this - I really don't want to prepare the template again. Should I just try adding more of it (for example 10x more, judging by the uncut band intensity) and treat it as digestion of 1 ug (so add only 1 ul of enzyme)?

If it is RNA, I would try as you suggest (the RNA shouldn't cause problems) but if your template is degraded, I would just prepare new template.  You may be able to transform cells with the prep if it is not too far gone, or maybe someone has glycerol stocks of bacteria with the plasmid and you can just grow some.  Preparing new template is easy (except the wait) and you can use miniprep DNA for most things you need to do, so no need to do a large scale purification.  Bad plasmid will cause you so many headaches trying to do cloning, that you will waste alot of time and figure out later you need to purify some new stuff anyway.  The good news is, that sounds like RNA bands to me!  Warren..

I see. Will try the RNase step and take it from there. I still find it slightly weird that I would have so much RNA there.. Is it a common problem with Qiagen preps?

Another related (pretty basic) question, as I know that XhoI doesnt cut well if site is methylated. If I wanted to get rid of methylation by growing plasmid again in Dam- bacteria, could I use the (methylated) plasmid I already have? Or do I have to go one step back to a non-methylated stock and use that for transformation?

Sorry to keep pestering you with questions! Thanks again for all your help..

All plasmid preps have a boatload of RNA if you do not get rid of it (digest it).  It's not unique to Qiagen.  Usually these kits (haven't used that particular one) come with RNase that is added to Solution I or at some point.  I wonder if the RNA might interfere with binding on the column though, which could reduce the plasmid yield.  I'd check -- I bet there is supposed to be an RNase step early on in the prep that may have been left out (intentionally or unintentionally).

Dam methylation doesn't effect XhoI, so growing in a dam- strain won't help anything.  There is a type of methylation that can effect XhoI (I forget what it is) but it is very context specific and usually the DNA around the XhoI site doesn't have the proper sequence.  I wouldn't worry about it, XhoI is a good enzyme.

But I do not think there is a problem transforming a methylated plasmid into a dam- strain (someone can correct me if I am wrong).  Just not necessary here.  Methylation problems don't come into play very often with regular plasmid cloning using common enzymes.  Warren..

[biobio]

Warren, on Sep 19 2009, 06:33 PM, said:

All plasmid preps have a boatload of RNA if you do not get rid of it (digest it).  It's not unique to Qiagen.  Usually these kits (haven't used that particular one) come with RNase that is added to Solution I or at some point.  I wonder if the RNA might interfere with binding on the column though, which could reduce the plasmid yield.  I'd check -- I bet there is supposed to be an RNase step early on in the prep that may have been left out (intentionally or unintentionally).

Dam methylation doesn't effect XhoI, so growing in a dam- strain won't help anything.  There is a type of methylation that can effect XhoI (I forget what it is) but it is very context specific and usually the DNA around the XhoI site doesn't have the proper sequence.  I wouldn't worry about it, XhoI is a good enzyme.

But I do not think there is a problem transforming a methylated plasmid into a dam- strain (someone can correct me if I am wrong).  Just not necessary here.  Methylation problems don't come into play very often with regular plasmid cloning using common enzymes.  Warren..

Yes.. there is RNase normally added to the lysis buffer - it could have gone off or something.. Will find out today or tomorrow.

Thanks for clearing up the specifics about XhoI methylation, I didn't quite get it before.

Another question that comes to mind: I amplified my insert using conventional Taq. I thought that because the gene is relatively small (~1.5kb), Taq would be enough. Do you agree or would you go for a high fidelity polymerase instead? (in which case, could I use the same thermal protocol for amplification?). I understand sequencing of the insert is of limited diagnostic use..

[Warren]

biobio, on Sep 20 2009, 07:37 AM, said:

Warren, on Sep 19 2009, 06:33 PM, said:

All plasmid preps have a boatload of RNA if you do not get rid of it (digest it).  It's not unique to Qiagen.  Usually these kits (haven't used that particular one) come with RNase that is added to Solution I or at some point.  I wonder if the RNA might interfere with binding on the column though, which could reduce the plasmid yield.  I'd check -- I bet there is supposed to be an RNase step early on in the prep that may have been left out (intentionally or unintentionally).

Dam methylation doesn't effect XhoI, so growing in a dam- strain won't help anything.  There is a type of methylation that can effect XhoI (I forget what it is) but it is very context specific and usually the DNA around the XhoI site doesn't have the proper sequence.  I wouldn't worry about it, XhoI is a good enzyme.

But I do not think there is a problem transforming a methylated plasmid into a dam- strain (someone can correct me if I am wrong).  Just not necessary here.  Methylation problems don't come into play very often with regular plasmid cloning using common enzymes.  Warren..

Yes.. there is RNase normally added to the lysis buffer - it could have gone off or something.. Will find out today or tomorrow.

Thanks for clearing up the specifics about XhoI methylation, I didn't quite get it before.

Another question that comes to mind: I amplified my insert using conventional Taq. I thought that because the gene is relatively small (~1.5kb), Taq would be enough. Do you agree or would you go for a high fidelity polymerase instead? (in which case, could I use the same thermal protocol for amplification?). I understand sequencing of the insert is of limited diagnostic use..

To me its a no-brainer if you are cloning -- use a high fidelity polymerase, the higher the better.  1.5 kb doesn't sound like much but it is a huge piece of DNA for taq to amplify with no mutations, and screening clones by sequencing is a real pain.  As for using the same cycling parameters, it depends on your polymerase -- if it was a simple PCR that you didn't have to tweak for taq to work, and you have "good" primers and template, there is a good chance the same parameters will work -- once again it depends on your polymerase though.  Warren..

[biobio]

Warren, on Sep 20 2009, 08:56 AM, said:

biobio, on Sep 20 2009, 07:37 AM, said:

Warren, on Sep 19 2009, 06:33 PM, said:

All plasmid preps have a boatload of RNA if you do not get rid of it (digest it).  It's not unique to Qiagen.  Usually these kits (haven't used that particular one) come with RNase that is added to Solution I or at some point.  I wonder if the RNA might interfere with binding on the column though, which could reduce the plasmid yield.  I'd check -- I bet there is supposed to be an RNase step early on in the prep that may have been left out (intentionally or unintentionally).

Dam methylation doesn't effect XhoI, so growing in a dam- strain won't help anything.  There is a type of methylation that can effect XhoI (I forget what it is) but it is very context specific and usually the DNA around the XhoI site doesn't have the proper sequence.  I wouldn't worry about it, XhoI is a good enzyme.

But I do not think there is a problem transforming a methylated plasmid into a dam- strain (someone can correct me if I am wrong).  Just not necessary here.  Methylation problems don't come into play very often with regular plasmid cloning using common enzymes.  Warren..

Yes.. there is RNase normally added to the lysis buffer - it could have gone off or something.. Will find out today or tomorrow.

Thanks for clearing up the specifics about XhoI methylation, I didn't quite get it before.

Another question that comes to mind: I amplified my insert using conventional Taq. I thought that because the gene is relatively small (~1.5kb), Taq would be enough. Do you agree or would you go for a high fidelity polymerase instead? (in which case, could I use the same thermal protocol for amplification?). I understand sequencing of the insert is of limited diagnostic use..

To me its a no-brainer if you are cloning -- use a high fidelity polymerase, the higher the better.  1.5 kb doesn't sound like much but it is a huge piece of DNA for taq to amplify with no mutations, and screening clones by sequencing is a real pain.  As for using the same cycling parameters, it depends on your polymerase -- if it was a simple PCR that you didn't have to tweak for taq to work, and you have "good" primers and template, there is a good chance the same parameters will work -- once again it depends on your polymerase though.  Warren..

Just to give the update..

I did the RNAse treatment on the plasmid and I saw a small reduction in the bottom band.. So it seems you were right, it was RNA..

In the meantime I went one step back and worked with an old mini-prep of the same plasmid. Cut it again and it all worked as it should.. Let's see I get colonies tomorrow!

Thanks a lot for your help!