Re: [ccp4bb] Off topic: denaturing urea gels

[Opher Gileadi]

In addition to the previous suggestions:

With very small gels, the sample composition and depth (in the well) have a 
strong effect on the resolution.
Rinse the wells with TBE buffer just before loading, as urea from the gel 
diffuses into the well and may prevent the sample from settling at the bottom. 
Minimize the amount of salt in the samples; try to load very small volumes (1-3 
ul), even if this means longer exposures later.

Re: [ccp4bb] Off topic: denaturing urea gels

[Zhijie Li]

Smith:
One should expect to see ladders each separated by 1 nt in such cases.

Mohammed:

My suggestions:
1) running at 70V seems low. I do not see a reason for not using higher 
voltages. 200Vx45min should be OK. IT is better to not let the BPB front run 
out of the gel - so that you know whether the DNA had all been chopped into 
single nts. (how do you visualize the bands? Is the oligo end-labelled?  EtBr 
should not work well for small fragments. But UV shadowing may work.)

2) boil the sample before loading (6-8 M urea should always be included in 
sample buffers). This is important because the enzyme might bind the DNA and 
slowly release them causing the smearing.

3) How does the uncleaved DNA alone (no enzyme at all) look on the same gel? If 
it is a sharp single band then you might need to pay extra attention to 
denaturing the samples before loading. If it is also smeary then it might be 
the gel system needs optimizing.

4) use 0.75mm gel. Thinner gels give better resolution. 

5) I found that DNA ran just fine in the discontinuous tris-glycine 
system(Laemmli). In other words,  the PAGE system routinely used for proteins 
(without SDS) works for DNA too, with the benefit of sharpening the bands.
 You may also consider experimenting with gel percentages(25%-30 % for example) 
so that you can see even single nts. The acry:bis ratio is not that important 
as long as you stick to one.


Zhijie



> On Sep 30, 2017, at 7:54 AM, Smith Liu  wrote:
> 
> your enzyme cannot give definite fragment. thus smear
> 
> 
> 发自网易邮箱大师
> 
> 在2017年09月30日 19:28，Mohammad Khan 写道：
> Dear all,
> 
> I am working with an exonuclease and I run the digested DNA on a 
> 8Murea-20%acrylamide gel in TBE buffer. I use the Mini-Protean BioRad system 
> and cast gels of about 8.6x6.5 cm dimensions with 1.5 mm thickness. I use a 
> 15 well comb. I run my gels at 70 V for as long as 4 hours till my undigested 
> DNA reaches half the gel distance. I use 20-30 nt long susbtrates.
> 
> I am mostly not able to get distinct bands of the digested products but 
> rather get a smear. Is there any way to make sure that I get distinct 
> digested products rather than a smear?
> 
> I am looking forward for suggestions from all!
> 
> Thank you.
> 
> Ciao!

Re: [ccp4bb] Off topic: denaturing urea gels

[Smith Liu]

your enzyme cannot give definite fragment. thus smear


发自网易邮箱大师


在2017年09月30日 19:28，Mohammad Khan 写道：
Dear all,


I am working with an exonuclease and I run the digested DNA on a 
8Murea-20%acrylamide gel in TBE buffer. I use the Mini-Protean BioRad system 
and cast gels of about 8.6x6.5 cm dimensions with 1.5 mm thickness. I use a 15 
well comb. I run my gels at 70 V for as long as 4 hours till my undigested DNA 
reaches half the gel distance. I use 20-30 nt long susbtrates.


I am mostly not able to get distinct bands of the digested products but rather 
get a smear. Is there any way to make sure that I get distinct digested 
products rather than a smear?


I am looking forward for suggestions from all!


Thank you.


Ciao!

[ccp4bb] Off topic: denaturing urea gels

[Mohammad Khan]

Dear all,

I am working with an exonuclease and I run the digested DNA on a
8Murea-20%acrylamide gel in TBE buffer. I use the Mini-Protean BioRad
system and cast gels of about 8.6x6.5 cm dimensions with 1.5 mm thickness.
I use a 15 well comb. I run my gels at 70 V for as long as 4 hours till my
undigested DNA reaches half the gel distance. I use 20-30 nt long
susbtrates.

I am mostly not able to get distinct bands of the digested products but
rather get a smear. Is there any way to make sure that I get distinct
digested products rather than a smear?

I am looking forward for suggestions from all!

Thank you.

Ciao!