Objective
- Re-do ligation with positive control
- Transform ligation and PCR from last week
- PCR of BSA
Bench work
- PCR of BSA (performed protocol from 5/26 again)
- 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
- DNA clean-up of pTXB1 samples after EcoRI (and phosphatase) digestion (Promega Wizard®, vacuum method)
- 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
- 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.
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AU CHEM-570 Lab Prep
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