SSB13Ntbk - Mar19
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Regular Zymo Cleanup
- Add 180 uL of Zymo ADB buffer to PCR reaction.
- Transfer into the Zymo column
- spin through, discard waste.
- Add 200 uL of Zymo Wash Buffer
- spin through, discard waste.
- Add 200 uL of Zymo Wash Buffer
- spin through, discard waste.
- spin for 90 seconds, full speed to dry.
- elute with water (50ul) into a fresh Eppendorf tube
EcoRI/BglII Digest of PCR Products
- Set up the following reaction:
8uL of eluted PCR product 1uL of NEB Buffer 2 0.5uL EcoRI 0.5uL BglII
- Incubate at 37 degrees on the thermocycler for 1hr
- Run an agarose gel, there was PCR product so the reaction was good
Zymo Gel Purification
- cut out bands minimizing extra gel matter.
- put in ependorf tube and add 600uL of Zymo ADB buffer (brown bottle).
- heat at 55, shake and/or vortex until the gel has dissolved.
- transfer into the Zymo column inside a collection tube (small clear guys)
- spin through, discard waste.
- Add 200 uL of Zymo Wash Buffer (which is basically 70% ethanol)
- spin through, discard waste.
- Add 200 uL of Zymo Wash Buffer
- spin through, discard waste.
- spin for 90 seconds, full speed to dry.
- elute with water (10ul) into a fresh Eppendorf tube, labeled "quiC4 EcoRI/BglII" and put in box with all the other aliquots