9 replies to this topic

[Julio-Claudian]

Dear all,

I have a questions regarding the amount of sample to load into a gel.

What is the usual amount people use when loading their DNA into the wells? Is it in the ng or ug region? I read 20ng but some sources on the net says 50 ug so do confirm this with me please.

Now, say I have quantitated (A260nm) my DNA and get 100ng/uL. Remember that I have to mix 1 uL of my sample (meaning I'd have 100 ng) into 4 uL loading dye. This would cause the amount to be diluted down by 5 times. Does this mean that the intensity pf the band when stained and visualized is that of 20 ng? I still feel 100 ng has been thrown inside the well despite the "dilution".

So, tell me. Does the rule of Chemistry (i.e. dilution and stuff) doesn't apply?

I asked because I did gel electrophoresis for my samples (new buffers, 5V/cm) but got nothing.

Thanks in advance!

[merlav]

If you add 100ng that's what you get, no matter how much you dilute (if you add all to the well).  I prefer to use aroud 75-100ng (up to 200ng) if I want to get a really nice band for a photo.

[rkay447]

For a really nice tight band you want to run in the ng range and 100 should be fine.  When I'm trying to purify DNA I'll run ugs but break it up over multiple wells or else it is just a smear on the gel.  

You took 100ng (1ul) and diluted it with 4ul dye.  If you load all 5ul, you just loaded all 100ng but if you only run 1ul of the DNA/dye dilution then you would be visualizing 20ng.  

How small is the piece of DNA you are wanting to see and how do you stain your gel or DNA?  EtBr runs TOWARD the negative (opposite the direction of DNA) so you can miss small peices of DNA without restaining the gel.  I always prefer to run the gel without EtBr and soak the gel in a solution afterward followed by destain in water.  Do you clearly see the marker at the size you want?

[Julio-Claudian]

rkay and merlav,

Thanks for the explanations. I got it crystal clear especially the amount to load as well as the calculation. Am visualizing both genomic DNA (extracted) as well as PCR products (1-8kb) <-- both on different gels though.

Got nothing (not even ladder) for the genomic part (today) so I thought it'd be good to ask about the amount for future works (PCR) too. The "nothingness" could be due to the buffer. Hmm..

Thanks people! Cheers!

[rkay447]

dreamchaser_jc, on Feb 3 2009, 08:30 AM, said:

rkay and merlav,

Thanks for the explanations. I got it crystal clear especially the amount to load as well as the calculation. Am visualizing both genomic DNA (extracted) as well as PCR products (1-8kb) <-- both on different gels though.

Got nothing (not even ladder) for the genomic part (today) so I thought it'd be good to ask about the amount for future works (PCR) too. The "nothingness" could be due to the buffer. Hmm..

Thanks people! Cheers!

If you can't even visualize the ladder then the gel is not appropriately stained with EtBr.  You need to put a couple ul of concentrated EtBr in a tub with TAE (the amount isn't critical but I usually use about 250ml).  Soak your gel in this solution for a few mins and then destain in water for about 15 mins.  If your gel really glows under UV you can continue destain until the bands stand out.  Perhaps you should be running a "positive" lane where you know there is DNA.  Just a ul of purified vector would work.  This staining procedure is especially important if you are trying to visualize something small.

[Trof]

Here we don't usually count ngs at all.

If I want to check my PCR product on gel I load either 10 ul (50ul reaction) or whole reaction (20ul). When I'm checking genomic DNA, purified product for sequencing or a plasmid, I use 1 ul of it. And the appropriate amount of ladder, usualy microliter too.

Our loading buffer is on the other hand 6x concentrated so we add 1 ul of it to 5 ul of sample. I found it a bit weird to have it the other way.

And we add yout EtBr into the gel when we make it, so it doesn't require any additional handling.

[hanming86]

Check back your staining procedure . I am suspecting something wrong with the staining since even the ladder ( positive control ) didn't work

Make fresh batch of staining solution ( 50 ng/ml ) and stain longer.

cheers

[Julio-Claudian]

Alright people! Thanks!

[TanyHark]

dreamchaser_jc, on Feb 4 2009, 08:28 PM, said:

Alright people! Thanks!

I think the minimum for viewing a DNA band on EtBr-agarose gel is about 20-25ng.

[perneseblue]

the minimum of EtBr about  10ng. You can see bands that low, but it is dependent on you getting sharp bands for that fragment size, and staining-destaining quickly to minimise band diffusion. Something that is not quite so easy.