Smolke:Protocols/Digests: Difference between revisions
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Adjust this mix depending on whether you're making a master mix. For one reaction, I up the BSA to 0.5uL (since I don't trust pipetting 0.2uL). For a master mix, you can judiciously reduce the amount of enzyme (<0.5uL/rxn) and adjust the relative enzyme amounts depending on the enzyme concentration (so use more an an enzyme at 5 U/uL and less of one at 20 U/uL). | Adjust this mix depending on whether you're making a master mix. For one reaction, I up the BSA to 0.5uL (since I don't trust pipetting 0.2uL). For a master mix, you can judiciously reduce the amount of enzyme (<0.5uL/rxn) and adjust the relative enzyme amounts depending on the enzyme concentration (so use more an an enzyme at 5 U/uL and less of one at 20 U/uL). | ||
Note: You can digest with some enzymes in as little as 1 hour, just make sure to use 1-2 ug of DNA max to help ensure that DNA is double cut.-SG | |||
==CIP== | ==CIP== |
Latest revision as of 21:23, 26 October 2011
Josh's double digest mix
Run overnight at 37C. Adjust this mix depending on whether you're making a master mix. For one reaction, I up the BSA to 0.5uL (since I don't trust pipetting 0.2uL). For a master mix, you can judiciously reduce the amount of enzyme (<0.5uL/rxn) and adjust the relative enzyme amounts depending on the enzyme concentration (so use more an an enzyme at 5 U/uL and less of one at 20 U/uL). Note: You can digest with some enzymes in as little as 1 hour, just make sure to use 1-2 ug of DNA max to help ensure that DNA is double cut.-SG CIP
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