Agarose Gel Electrophoresis

[@nwalk4529]

great video thank you!

[@LeeFarmberg]

Thank you so much for your video!

[@biochemfusion]

Fabulous respected sir

[@ProfessorDrewCollop]

So nice of you. Thanks

[@VedikaAnkitaAglawe-g2x]

Thanks a lot for replying

[@maleeshapriyanjana7604]

very good explanation!

[@sonu0791]

Good practical video

[@NKD_MaDlamini]

Sooo cool

[@nombekosikhosana9233]

What’s that filter you used to see the DNA bands?

[@ProfessorDrewCollop]

In the old days we used ethidium bromide to stain the DNA and a UV light to cause the ethidium bromide to fluoresce. Both ethidium bromide and UV are somewhat dangerous. We now us a chemical call Red-Safe to stain the DNA. This does not require UV light. Instead we use a blue light and an orange filter to visualized the nucleotide bands.

[@pleasuregaming2601]

What was the amount and ratio used in loading dye : DNA

[@ProfessorDrewCollop]

We usually use 6X DNA Loading Dye. We mix 5 uL of DNA with 1 uL of 6X Loading Dye. Final volume is 6 uL, resulting in 1X concentration of the Dye. Hope this makes sense.

[@reynaguillenenriquez6186]

How could I do the 6X? And do you recommend use 3 ul of DNA and 7ul of 6X? I have watched it some time...

[@ProfessorDrewCollop]

Please see the previous comment "We usually use 6X DNA Loading Dye. We mix 5 uL of DNA with 1 uL of 6X Loading Dye. Final volume is 6 uL, resulting in 1X concentration of the Dye. Hope this makes sense." 6x loading dye means it is 6 times too concentrated. You need to dilute it 1/6 so it is 1x loading dye. 1 uL dye + 5 uL DNA = 6 uL total solution. 1 uL dye / 6 uL total solution = 1/6

[@VedikaAnkitaAglawe-g2x]

What is x here ?

[@ProfessorDrewCollop]

x = times; therefore 6x loading dye = 6 times loading dye. This meaning it is 6 times more concentrated then the working stock. I is made this way to mix 5 uL of DNA with every 1 uL of 6x loading dye. At this dilution (1/6) it will be 1x loading dye. Hope this helps.

[@mad1cajon]

Can I ask what is the name of the filter? Orange 21?

[@ProfessorDrewCollop]

Not exactly sure the name of the filter. It is the amber filter that came with the IO Rodeo Large Blue LED Transilluminator. I believe the blue light is at 470 nm and the amber filters light to about 580 nm.

[@mad1cajon]

@@ProfessorDrewCollop Thank you, much appreciated.

[@harisonsang3800]

What's TAE?

[@ProfessorDrewCollop]

TAE is an acronym. T = Tris, A = Acetic Acid, E = EDTA. Tris is a strong base. It has as very high pH. The Acetic Acid is a weak acid. It has a low pH. Mix them together and it makes a buffer. A buffer stabilizes the pH, so it cannot go up too much or down too much. EDTA is a chemical that chelates divalent cations such as Mg^+2. Chelate = Binds to them so they cannot bind to anything else. Mg^+2 is a necessary cofactor for DNase, an enzyme that degrades DNA. No Mg^+2 = No DNase activity = Higher concentrations of DNA. Running electricity in water leads to electrolysis of H2O. H2O --> H+ & OH- The H+ ions can interact with the negatively charge DNA molecule and neutralize it. If the DNA is neutralized, it will not be pulled through the gel to the positive terminal. The buffer will bind to the H+ and OH- ions and buffer them, so they will not interact with the DNA.