DNA Extraction for Ladder Assay - Protocols to get best Agarose gels (Jul/12/2007 )
I am doing DNA isolation by phenol chloroform ext method. then i am precipitating by ethanol overnight. i get a reasonable good DNA. then i am dissolving in TE, but the sample is viscous and difficult to load into gels. how to reduce viscosity, so that sample loads in wells easily.
i hope any one of you may help/ suggest at your earliest.
Can any one tell that, can we take break in protocol of DNA isolation/purification/extraction.....by freezing the samples in between steps. will it going to harm the yield/beauty of DNA isolated.
also tell me some tips to beutify gels.....to avoide smear...etc.
i would be very thankful if any one rushes his remarks or refer me some site/link/person.
If the sample to be loading in gel is to viscous, that means you have lots of dna and should add more TE to dissolve it. When dissolving DNA incubate at 37-40C to help it to inter in solution. Before loading the gel did you check the DNA concentration????. Always check the concentration before begin any assay, never estimate quantity that's a very common error. the thing is no to add for example 2 micro, but to know how much you are adding (eg 1 micro, 200 nano etc.). When knowing concentration will avoid smears. I had never stored the samples in between the process, but I sugest is better to organize the workand make a schedule. If you don't have time for a procedure is better to leave it for othe time, mking things with hurry can introduce mistakes, so you have to repeat again, lossing time and money in the meanwhile.
If your DNA is viscous, may be it’s too concentrate, dilute with TE 1X. Or it could be not complete dissolved. Incubate few minutes at 40 or 50°C and mix after adding TE.
Add SDS to the loading buffer. It’s really good to avoid viscosity and smear.
You can freeze the pellet before the extraction for days without problem.
Don’t stop in the sol I, II, III steps.
Don’t overdry the DNA pellet.
- Precipitating with isopropanol without overnight incubation (to save time and have less salts concentrations).
- A thin gel looks much better.
- To avoid smear and have better separation use new TAE or TBE and run at 80-100 volts (although, some times I have beautiful gels ran at 250 V) .
- Don’t load lot of DNA.
Dilution might just work if it is too viscous. For precipitation process, you can leave your sample in 100% alcohol for overnight precipitation will be alright.
And yea.. for smearing, I do agree with aztecan. Don't load lotsa of DNA. Or perhaps.. degradation might happen that lead to smearing. Cheers