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
 * info from http://avery.rutgers.edu/WSSP/StudentScholars/project/archives/onions/runagel.html

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

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.

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.

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"

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

DNA gel extraction