Team:PKU Beijing/Notebook/Protocol/DNA Gel Extraction
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Here is a suggested protocol; this protocol can be used to purify a wide range of DNA fragments with recoveries of >80%. The bolded should be noticed for a nice DNA extraction. | Here is a suggested protocol; this protocol can be used to purify a wide range of DNA fragments with recoveries of >80%. The bolded should be noticed for a nice DNA extraction. | ||
- | 1. Excise gel slice containing the DNA fragment using a clean scalpel or razor blade. ''Cut as close to the DNA as possible to minimize the gel volume''. Place the gel slice into a pre-weighed 1.5 ml tube and weigh. Record the weight of the gel slice. <br> | + | 1. Excise gel slice containing the DNA fragment using a clean scalpel or razor blade. '''Cut as close to the DNA as possible to minimize the gel volume'''. Place the gel slice into a pre-weighed 1.5 ml tube and weigh. Record the weight of the gel slice. <br> |
- | 2. Put EB (elution buffer) at ''65 degree'' water bathing.<br> | + | 2. Put EB (elution buffer) at '''65 degree''' water bathing.<br> |
- | 3. Add a 3:1 volume of Solution Buffer to the gel slice (volume:weight) (e.g., add 300 ul of Binding Buffer for every 100 mg of agarose gel). Incubate the gel mixture at 60 degree for 5 min at least ''until the gel slice is completely dissolved''. Mix the tube by inversion every few minutes to facilitate the melting process. ''Check the color of the solution''. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 ul of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow.<br> | + | 3. Add a 3:1 volume of Solution Buffer to the gel slice (volume:weight) (e.g., add 300 ul of Binding Buffer for every 100 mg of agarose gel). Incubate the gel mixture at 60 degree for 5 min at least '''until the gel slice is completely dissolved'''. Mix the tube by inversion every few minutes to facilitate the melting process. '''Check the color of the solution'''. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 ul of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow.<br> |
- | 4. Pour the solution to a fresh adsorption column. Centrifuge at 13000rpm for 1 min. Pour off the liquid in the collection tube. ''For critical samples'', repeat the operation above.<br> | + | 4. Pour the solution to a fresh adsorption column. Centrifuge at 13000rpm for 1 min. Pour off the liquid in the collection tube. '''For critical samples''', repeat the operation above.<br> |
5. Add 600 ul washing buffer (WB) before centrifugation at 13000 rpm for 1 min. Pour off the liquid into beaker. <br> | 5. Add 600 ul washing buffer (WB) before centrifugation at 13000 rpm for 1 min. Pour off the liquid into beaker. <br> | ||
- | 6. ''Centrifuge at 13000rpm for 10 min'' to spin the ethanol down. <br> | + | 6. '''Centrifuge at 13000rpm for 10 min''' to spin the ethanol down. <br> |
- | 7. Put the column into a fresh EP tube. If necessary air-dry the pellet for 10-15 min to avoid the presence residual ethanol in the purified DNA solution. ''Residual of ethanol in the DNA sample may inhibit downstream enzymatic reactions''.<br> | + | 7. Put the column into a fresh EP tube. If necessary air-dry the pellet for 10-15 min to avoid the presence residual ethanol in the purified DNA solution. '''Residual of ethanol in the DNA sample may inhibit downstream enzymatic reactions'''.<br> |
8. Add 30-50 ul elution buffer (EB) to elute the DNA. <br> | 8. Add 30-50 ul elution buffer (EB) to elute the DNA. <br> | ||
9. Get 5 ul of the eluted sample to identify with electrophoresis. | 9. Get 5 ul of the eluted sample to identify with electrophoresis. |
Revision as of 07:50, 7 October 2009