Information about DNA Gels

2015-01-13

azim58 - Information about DNA Gels

see Optimal Gel Electropheresis Protocol 2-14-13

Best percent gel for 1 kb bands -> 0.8%
100 bp -> 1.5%
4-6kb vector -> 0.6%

What voltage should be applied to a 0.8% gel?

http://www.ncbe.reading.ac.uk/ncbe/protocols/DNA/PDF/DNA13.pdf

For the best resolution, 0.8% agarose gels should be run at no more than
5 V/cm.
Note that only about 80% of the voltage on the power source is applied to
the gel. The other 20% is dissipated in the buffer.

Length of "medium 100 mL" gels: 10.9 cm

y*0.8/(10.9*cm) = 5 V/cm -> y = 68.13 V

Length of "large 350 mL" gels: 27.5 cm

y*0.8/(27.5*"cm") = 5*"V"/"cm" -> y = 172 V

What's the lowest amount of DNA that is visible?

It fluoresces under UV light when intercalated into the major groove of
DNA (or RNA). By running DNA through an EtBr-treated gel and visualizing
it with UV light, any band containing more than ~20 ng DNA becomes
distinctly visible.
Source:
http://en.wikipedia.org/wiki/Agarose_gel_electrophoresis

So about 20 ng

How long should a gel be stained with ethidium bromide?

* 20-30 minutes with a 0.5 ug/mL solution or overnight at 4 C
  o info from
    http://avery.rutgers.edu/WSSP/StudentScholars/project/archives/onions/r
    unagel.html

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Large gel (350 mL gels)
How many samples on one comb?
We have 8 combs that can fit 24 samples, 6 that can fit 23, and 4 that
can fit 20
8*24 = 192
6*23 = 138
4*20 = 80
192+138+80 = 410

Some of the cases can fit about 5 combs. Maximum samples with two cases
and 5 combs each?
8*24+2*23 = 238

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When running a gel, it may be wise to load approximately the same amount
of sample as the amount of the ladder since the amount of DNA can affect
the migration rate. Also sometimes running the gel at a cold temperature
may be a good idea since overheating can cause samples at the edges of
the gel to run at a different rate than samples in the middle of the gel.

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Gels can be left in the 4 C cold room in buffer with aluminum foil
covering the gel to prevent degradation of the ethidium bromide for
several days, and the resulting image will still look good.  Evidence for
this on 8-28-13 experiment.

Gels can be left in the 4 C cold room in buffer overnight. In fact,
images from these gels look quite good. Example image can be seen on
experiment performed on 2-27-13.
"S:\Research\Cancer_Eradication\Discovering tumor specific antigens\Tumor
cDNA Library\2-27-13 4 C overnight test\4C overnight gel test 2hr 70 V
1p5 exp 2-27-13.jpg"

Can I use a gel that sat in 4 c room (without any buffer to keep the gel
wet and without any ceram wrap to prevent the gel from drying) over
weekend or overnight?
-The gel is somewhat usable, but the bands are less sharp and more
crooked. I suspect that this is due to uneven drying of the gel which
causes the gel to have different densities in different areas. An image
comparing a fresh gel and a gel left in the 4 C room over the weekend can
be found here:
--"C:\kurt\storage\CIM Research Folder\DR\2012\10-29-12\comparison of
fresh and over weekend gel 10-29-12.pdf"
-Also a gel that has been moved back and forth from 4C to RT in buffer
for several days seems to result in blurry and crooked bands. I have one
example of this on 6-25-13 unless this result was caused by something in
the buffer or something else.

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Preparing DNA ladder stock portal 11-1-12
Preparing 6X dye portal 11-1-12

DNA gel extraction

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