Question

I've got a DNA agarose gel electrophoresis question
The well of an agarose gel can fit 30 µl of sample.
I would like to prepare 50 ng of DNA sample that has a concentration of 10 ng/µl. I've been provided with sterile water and 6X concentrated gel loading buffer. How much of each component (DNA, water and 6X gel loading buffer) should I mix to end up with 50 ng of DNA in a total sample volume of 30 µl?
The volume i got for DNA is 5 µl but i dunno how to proceed for the gel loading buffer. Anyone who performed this kinda gel electrophoresis to answer me the proportion of buffer to add?

Answer 1

i'm kinda confused on what you want. Do you have a solution for DNA? because what you wrote ("ng") is not a concentration. So i'm not sure how you arrived at 5 \(\mu L\) of DNA.

Answer 2

I've been provided with 10 ng/µl of DNA (that's a concentration).... I want 50 ng of DNA.
So i thought that if 10 ng fits in 1 µl, then 50 ng would fit in [(50 X 1)/10] = 5 µl of DNA

Now i want to know about the buffer. How much of the buffer do i need? Bear in mind that in the well, i'm allowed to put a total of 30 µl of sample (DNA+Buffer+Water).

Answer 3

do you know the final concentration of DNA in the solution, or simply that you need 50 ng?

Answer 4

I just need 50 ng. my objective is to obtain 5ng of DNA out of 10ng/µl (provided to me).

Answer 5

okay, so you got 5 \(\mu L\) of DNA solution and you need 30 \(\mu L\).

so 30 -5 \(\mu L\)= 25 \(\mu L\); do you know the proportions of water and buffer you're using?

Answer 6

Yes i have 25 µl remaining.
No, i dont know the proportions of water and buffer, not mentioned in the question. But it says that the process requires the buffer to be concentrate. Honestly, i find that smething is missing in the question i got.

Answer 7

i would just add the rest (25 \(\mu L\)) buffer. Buffers are already set at a pH and if you add water it changes the pH. I've ran a few gels and i've never added water (unless you're making the buffer from scratch).

Answer 8

so u suggest me to do this:
To 25 µl of buffer, add 5µl of DNA sample?
I've not yet performed this in the laboratory, will do so soon ^_^

Answer 9

yes. hold on a min, let me check my past lab manual

Answer 10

Seems logical what you told me though. okay i'll wait. ^_^

Answer 11

um what i've done is a little different, I made the buffers from scratch, but no water was added after doing that. So i would go with what i said previously.

Answer 12

Yeah it seems wise what u said because yeah if you already prepared the buffer at a certain pH, further adding water (if u added that while preparing the buffer) would definitely lower it, ryt?

Answer 13

... and thank you for all your help aaron, i appreciate =)

Answer 14

yeah, you use the buffer as the main solution to dilute anything further. Never water, biological molecules and pure water don't mix well, you need salts. No problem, good luck with the gel !

Answer 15

I still have no practical experience with preparing buffers & gel electrophoresis yet, will do so in the coming weeks. I'll bear in mind what you said. And thank you again, take care =)