SSB13Ntbk - Mar19

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Regular Zymo Cleanup

  1. Add 180 uL of Zymo ADB buffer to PCR reaction.
  2. Transfer into the Zymo column
  3. spin through, discard waste.
  4. Add 200 uL of Zymo Wash Buffer
  5. spin through, discard waste.
  6. Add 200 uL of Zymo Wash Buffer
  7. spin through, discard waste.
  8. spin for 90 seconds, full speed to dry.
  9. 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

  1. cut out bands minimizing extra gel matter.
  2. put in ependorf tube and add 600uL of Zymo ADB buffer (brown bottle).
  3. heat at 55, shake and/or vortex until the gel has dissolved.
  4. transfer into the Zymo column inside a collection tube (small clear guys)
  5. spin through, discard waste.
  6. Add 200 uL of Zymo Wash Buffer (which is basically 70% ethanol)
  7. spin through, discard waste.
  8. Add 200 uL of Zymo Wash Buffer
  9. spin through, discard waste.
  10. spin for 90 seconds, full speed to dry.
  11. elute with water (10ul) into a fresh Eppendorf tube, labeled "quiC4 EcoRI/BglII" and put in box with all the other aliquots
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