User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/07/25

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Objective

  1. Re-do ligation with positive control
  2. Transform ligation and PCR from last week
  3. PCR of BSA

Bench work

  1. PCR of BSA (performed protocol from 5/26 again)
  2. Digestion of pTXB1 with EcoRI
    • 30μL dH2O + 5μL NEB2 + 10μL pTXB1 (from 6/16) + 5μL EcoRI (2kU/mL)
    • 2hr @ 37°C
    • Added phosphatase in final 15 minutes of digestion
  3. DNA clean-up of pTXB1 samples after EcoRI (and phosphatase) digestion (Promega Wizard®, vacuum method)
  4. Analytical minigel:
    • Lane 2: ladder
    • Lane 4: pTXB1 EcoRI digest + phosphatase
    • Lane 5: pTXB1 EcoRI digest
    • Lane 7: QuikChange pTXB1.His + BSA from 7/21
    • Lane 8: QuikChange pTXB1.His + BSA (-) control from 7/21
    • Lane 10: Ligation ddpTXB1.His + ddBSA from 7/21
    • Lane 11: Ligation ddpTXB1 + ddBSA from 7/21
    • Lane 13: pTXB1 miniprep from 6/16

Results

Gel

Analytical gel 07252011labeled.jpg

  • The gel displayed only the contents of Lane 13 -- the pTXB1 positive control. The EcoRI digests contained too much alcohol from purification and did not stay in the lanes.