Promegas' Wizard® PCR Preps DNA Purification System; Purification Without a Vacuum Manifold (Using 3ml Luer-Lok® Syringes) is the kit i am using. I am purifying pcr products from a 1.5x TAE gel. I think the issue is during the washing part with Isopropanol and that it is not being washed off the column all the way and when I elute my DNA sample there is still isopropanol in my sample causing them to not stay into the wells of the gel and evaporate out. I have tried to spin the columns twice at 16,000xg for two minutes and let air dry for a minute before elution. Any advice or ideas? My next step is DNA sequencing (In some of my samples I have two bands per sample so I would like to purify from a gel)
you can try adding more loading dye to the samples, Also run a loading dye only sample with water instead of your dna sample to check that the loading dye is working correctly
Thanks for the suggestions! I have already tried adding more loading dye to the samples. I've also tried a 5x loading dye and 10x and had no luck with both of them. But those loading dyes work well with other samples so I don't think that is the problem.
Following few tips might help.
Are you eluting in water or TE? elution in TE buffer might help.
How are you sure that the isopropanol is causing the samples to float out of well? Have you checked the 260/280 ratio? It would be really high if you have isopropanol.
You can add 30% glycerol in sample to sink samples into well.
This happens sometimes ,unfortunately, it just because the ethanol or the isopropanol of the washing solution, the best thing to do is to spin down the tube with the lid open in the vacuum centrifuge until total evaporation of the elution solution and then elute it in MiliQ water or your elution buffer a second time. After that everything will be OK. Hope that my answer will be useful for you.
Hello, Also besides all above make sure you running buffer has the proper pH.
you should proper evaporate isopropanol before elution step.
to purify your sample you can use DNA gel extraction kits to extract the the band which in right molecular weigh from the agarose gel and next you can make nurse or second pcr by using the band which was extracted from the gel as a templet to increase the concentration of your product.
If you have residual alcohol in your samples you will get inhibition problems in your downstream sequencing
make sure your intensity of band should be strong enough to get maximun concentration of final elute so that it will resemble further with gel electrophoresis. you can try by loading final elute 2,4,6 ul by adding 6X dye. may be you will get result.
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