Needs some expert advice in purifying a 10,300 kb fragment (after restriction) from agarose gel? I am getting very low yield, only few ngs!!! whereas my starting concentrations is more than 5 ugs. Any suggestions!
http://www.pall.in/main/laboratory/literature-library-details.page?id=35488
Try using lower agarose conc. gels (~ 0.8%) when running your sample. Secondly, try using the gel extraction kit from Qiagen. Have used it myself.
it would be good to see a picture of your gel Anamika in case your template DNA is only partially cut so you may be losing product in the high molecular weight poorly separated region. Is this cut from a plasmid. You should use low gelling agarose and if you are getting low yields try adding a co-precipitant such as glycogen to bring down a better precipitate. It will not increase the OD260 but helps precipitation. tell us your template and cutting time,temperature and enzymes please. Is it possible that you have mixed colonies growing only some of which have the insert so there may not be much insert in your 5ug of starting vector DNA?
Hi Anamika,
I used to purify my digestion product using DNA purification kit. You can use PCR purification kit, However you need to check if its suitable for restriction reaction, GeneAll have a kit for (PCR, Digestion) DNA purification. so double check with your brand as some column has a specific fibre matrix for one purpose only.
You can use innuPREP Gel Extraction Kit. The general procedure for DNA extraction: gel solubilization, binding of DNA onto spin filter (green), washing of the bound DNA and elution of DNA
i am sure gel extraction kit for high mol wt DNA works well . Try Zymoclean™ Gel DNA Recovery Kit (for DNA 11 kb to 23 kb the recovery is 50-70%.)
thank you everyone for your advices. I am using HpaI and BstBI from NEB for cutting my plasmid in a serial digestion for 90 mins at 37degs and then 20mins at 65 degs(as BstBI is a time saver enzyme). The product was two bands of correct sizes, so I am sure the digestion is not a problem. The bands were very bright which I tried to purify using two different kits, from Qiagen and from Thermoscientific, but result was same. So finally, I just amplified my fragment of interest using Phusion polymerase, instead of wasting lot of time behind something that didn't want to work!!! Thanks again :)
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