partial digestion problem - (Jan/18/2006 )

Hi,
I have a very low-concentrated DNA (29 ug/ml), and I'm trying to do partial digestion with Sau3AI. SO far I tried different incubation times (10-50 min at 37C), and different DNA concentration, but nothing worked. I am not sure now if it is possible to do partial digestion at all.
Can anyone help me with this?
Thanks!

Hi Yata,

you don't need to try different DNA concentration, but different concentrations of the enzyme. Keep the DNA concentration steady and consider this:

"One unit is defined as the amount of enzyme required to digest 1 µg of λ DNA in 1 hour at 37°C in a total reaction volume of 50 µl"


Your concentration is 29ng/ul ie 1/40th of 1ug, so I would start with 1U of enzyme, 0.1U, 0.05U, 0.025U and 0.01U. Keep the incubation time at around 20-30 minutes, set up the samples on ice and immediatly inactivate the enzyme after incubation with 20min at 65degrees.

You can also try using NEB buffer 2 instead of the one recommeded, as the enzyme supposedly only has 50% efficiency in that buffer.


Hope that helps

LeserattePD

When did partial digestion some time ago, I had a lot more DNA to spend (why not do an extra prep, or is this impossible?) and then I cut with serial dilutions of my restriction enzyme for no longer than 15 minutes and heat inactivated it. (To make sure it is inactivated as soon as possible, raise temperature above 65, it will not damage your DNA).

You can be sure that partial digest works, it's just a pain to do, but when you got it working you feel really well (I know I did).

Hope this helps.

Thanks a lot! I'll try that!
I think I only tried 0.5U for 0.58ug of DNA (since 1u-for 1 ug DNA).
Another question - how to resolve digested products - I tried 1%Agarose in 1xTAE, and ran on 150V for ~30min, but I didn't see anything. I know some people do sucrose gradient, but I 've never done this, and we don't have the equipment for that in our lab.
Any suggestions?
Thanks again!

[quote name='yata' date='Jan 22 2006, 08:12 PM' post='38005']
Another question - how to resolve digested products - I tried 1%Agarose in 1xTAE, and ran on 150V for ~30min, but I didn't see anything. I know some people do sucrose gradient, but I 've never done this, and we don't have the equipment for that in our lab.
Any suggestions?



Hi

I assume you want to do a partial digestion is because SauI restriction site is also present in your gene of interest, is that correct?
maybe is for a different reason/assay?

Well, if it is for cloning porpuses, to know what kind of gel you need I need to know what are the expected DNA band sizes?

This is for genomic library construction. I'm doing partial digestion to cleave randomly genomic DNA. I expect it to be 3-10 kb, since it is random , but no less than 3kb.


[quote name='macedo' date='Jan 22 2006, 02:07 PM' post='38010']
[quote name='yata' date='Jan 22 2006, 08:12 PM' post='38005']
Another question - how to resolve digested products - I tried 1%Agarose in 1xTAE, and ran on 150V for ~30min, but I didn't see anything. I know some people do sucrose gradient, but I 've never done this, and we don't have the equipment for that in our lab.
Any suggestions?



Hi

I assume you want to do a partial digestion is because SauI restriction site is also present in your gene of interest, is that correct?
maybe is for a different reason/assay?

Well, if it is for cloning porpuses, to know what kind of gel you need I need to know what are the expected DNA band sizes?
[/quote]

Oh I see,
I cannot help you on that as I never made a genomic DNA library myself. We bought one already made
Good luck

The general rule with agarose is, that the smaller the band, the more percentage of agarose you use (1% is good for around 1-2kb bands). 3-10kb is quite large, therefore you'll need to use 0.5% Agarose gels and run them up to 3h. If you have EtBr in the gel and buffer, you can simply take out the gel to have a look and then put it back. The voltage is really dependent on the distance between the electrodes (there's some formula regarding this, but can't remember off the top of my head). If you use a low percentage agarose gel, you can raise the voltage. For high percentage gels you have to lower the voltage otherwise they melt.

LeserattePD


[quote name='yata' date='Jan 22 2006, 09:50 PM' post='38030']
This is for genomic library construction. I'm doing partial digestion to cleave randomly genomic DNA. I expect it to be 3-10 kb, since it is random , but no less than 3kb.


[quote name='macedo' date='Jan 22 2006, 02:07 PM' post='38010']
[quote name='yata' date='Jan 22 2006, 08:12 PM' post='38005']
Another question - how to resolve digested products - I tried 1%Agarose in 1xTAE, and ran on 150V for ~30min, but I didn't see anything. I know some people do sucrose gradient, but I 've never done this, and we don't have the equipment for that in our lab.
Any suggestions?



Hi

I assume you want to do a partial digestion is because SauI restriction site is also present in your gene of interest, is that correct?
maybe is for a different reason/assay?

Well, if it is for cloning porpuses, to know what kind of gel you need I need to know what are the expected DNA band sizes?
[/quote]
[/quote]

You will likely have a hard time visualizing your DNA on a gel, given the low concentration and the fact that it is genomic DNA. Since there will be no sharp bands, the DNA will spread out over the entire gel, and the bands will be very diffuse. With low concentrations, they will likely be invisible. Can you prepare more genomic DNA? Can you concentrate the DNA you have? To see a sharp band, you need about 20 ng of DNA. With diffuse bands, I would guess you need to load 500 - 1000 ng of DNA in a well. You might also double check that the genomic DNA is cut by Sau3AI. My organism, e.g., is resistant due to methylation.