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Team:IIT Madras/Notebook/Protocols/Gel elusion
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Revision as of 10:02, 2 October 2011
IIT Madras
And then the E.coli said, "Let there be light"
Gel Elusion
- Prepare a 0.8% low melthing agarose gel.
- Add 50 ul preparative reaction product into large wells.
- Run the gel at 100 V for 30 – 45 mins.
- Cut the gel with restricted DNA and keep it in an microfuge tube.
- To the cut DNA, add 3 volumes of Chaotropic salt. Incubate at 50 C for 10 minutes.
- Add 10 ul of GPS (Glass Powder Solution) for a PCR product and 15 ul for a vector and incubate at Room Temperature for 10 mins. Shake the tubes continuously so that GPS is in suspension and doesn’t settle down.
- Centrifuge the tubes at 12000 rpm for 2 minutes and discard supernatant.
- Wash pellet by adding 50 volumes of wash buffer. That is, 500 ul of Wash buffer for insert and 750 ul for vector.
- Resuspend the pellet in 20 ul of water and heat at 50 C for 10 minutes. Centrifuge the tubes at 12000 rpm for 2 mins. Store supernatant at -20 C.
