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| | <noinclude>{{:Team:EPF-Lausanne/Template/Header}}</noinclude> | | <noinclude>{{:Team:EPF-Lausanne/Template/Header}}</noinclude> |
| | {{:Team:EPF-Lausanne/Template/ProtocolHeader|Restriction site digestion|{{{1|}}}}} | | {{:Team:EPF-Lausanne/Template/ProtocolHeader|Restriction site digestion|{{{1|}}}}} |
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| - | <!-- Insert content here (don't forget to set the title up here!) -->
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| - | <!-- Don't forget to add this protocol to the protocol list page: --> | + | <noinclude>{{:Team:EPF-Lausanne/Template/SetTitle|Restriction Site Digestion}}</noinclude> |
| - | <!-- https://2012.igem.org/Team:EPF-Lausanne/Protocol -->
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| | # Look for the best pair of restriction sites, ideally with similar digestion temperatures and times. | | # Look for the best pair of restriction sites, ideally with similar digestion temperatures and times. |
| - | # Get from the fridge: | + | ## [http://tools.neb.com/NEBcutter2/ NEBcutter] for finding cutting enzymes. |
| - | ## Buffer x10 | + | ## [http://www.neb.com/nebecomm/DoubleDigestCalculator.asp Double Digest Finder] for the parameters. |
| - | ## BSA | + | # Calculate the amounts required of: |
| | + | ## DNA |
| | + | ## Buffer (usually from 10x to 1x) |
| | + | ## BSA, if needed (usually from 100x to 1x) |
| | + | ## Enzymes (depends on the amount of DNA) |
| | + | ## Water |
| | + | # Get the recommended buffer (and BSA if needed) from the freezer and let defreeze. |
| | + | # Mix all the ingredients, except DNA, in a tube. |
| | + | # Note: Enzymes should stay no longer than a couple of minutes out of the freezer. Don't touch the bottom of the tubes! Don't vortex! |
| | + | # Distribute the mix in as many tubes as DNA samples and add the DNA. |
| | + | # Keep in the Thermomixer at the recommended temperature. |
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| | + | Sowmya's recommended amounts (50 µl total solution): |
| | + | * 5 µl of 10x buffer |
| | + | * 0.5 µl of 100x BSA |
| | + | * 1 µl of each enzyme |
| | + | * 5 µl of DNA |
| | + | * 37.5 (up to 50 µl) of water. |
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| | + | Protocol based on what was done on [[Team:EPF-Lausanne/Notebook/4_July_2012 | July the 4th]]. |
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| | {{:Team:EPF-Lausanne/Template/ProtocolFooter}} | | {{:Team:EPF-Lausanne/Template/ProtocolFooter}} |
| | <noinclude>{{:Team:EPF-Lausanne/Template/Footer}}</noinclude> | | <noinclude>{{:Team:EPF-Lausanne/Template/Footer}}</noinclude> |
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