SBB13Ntbk - Mar14

From OpenWetWare

Jump to: navigation, search

The PCA amplification did not yield enough PCR product, so need to do re-amplification.

  1. Ran an analytical gel, 1%, 10ul of PCR product. Thankfully saw PCR product, but a very small amount.

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

Re-Amplification

  1. 1 ul each outer oligo (10 uM)
  2. 1 ul purified pca product
  3. .5 ul phusion
  4. 10 ul 5x phusion buffer
  5. 5 ul 2mM dNTPs
  6. 32.5 ul H2O

NOTE: I accidentally used the wrong protocol for Re-Amplication.

Personal tools