Team:PKU Beijing/Notebook/Protocol/DNA Gel Extraction
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(New page: {{PKU_Beijing/Header}} {{PKU_Beijing/Sidebar_Notebook}} {{PKU_Beijing/Header2}} ==='''DNA Gel extraction protocol'''=== download PDF version ...) |
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{{PKU_Beijing/Sidebar_Notebook}} | {{PKU_Beijing/Sidebar_Notebook}} | ||
{{PKU_Beijing/Header2}} | {{PKU_Beijing/Header2}} | ||
+ | [[Team:PKU_Beijing/Notebook|Notebook]] > [[Team:PKU_Beijing/Notebook/Protocol|Protocol]] > [[Team:PKU_Beijing/Notebook/Protocol/DNA_Gel_Extraction|DNA Gel Extraction]] | ||
==='''DNA Gel extraction protocol'''=== | ==='''DNA Gel extraction protocol'''=== | ||
- | [[Media:PKU_DNA_Gel_extraction_protocol.pdf|download PDF version]] | + | <html><img src="https://static.igem.org/mediawiki/2009/9/91/PKU_Adobe_Reader_Logo.jpg" width=20></html>[[Media:PKU_DNA_Gel_extraction_protocol.pdf|download PDF version]] |
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. | + | 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. |
- | 2. Put EB (elution buffer) at '''65 degree''' water bathing. | + | |
- | 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. | + | 2. Put EB (elution buffer) at '''65 degree''' water bathing. |
- | 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. | + | |
- | 5. Add 600 ul washing buffer (WB) before centrifugation at 13000 rpm for 1 min. Pour off the liquid into beaker. | + | 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. |
- | 6. '''Centrifuge at 13000rpm for 10 min''' to spin the ethanol down. | + | |
- | 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'''. | + | 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. |
- | 8. Add 30-50 ul elution buffer (EB) to elute the DNA. | + | |
+ | 5. Add 600 ul washing buffer (WB) before centrifugation at 13000 rpm for 1 min. Pour off the liquid into beaker. | ||
+ | |||
+ | 6. '''Centrifuge at 13000rpm for 10 min''' to spin the ethanol down. | ||
+ | |||
+ | 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'''. | ||
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+ | 8. Add 30-50 ul elution buffer (EB) to elute the DNA. | ||
+ | |||
9. Get 5 ul of the eluted sample to identify with electrophoresis. | 9. Get 5 ul of the eluted sample to identify with electrophoresis. | ||
Latest revision as of 10:04, 9 October 2009
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