Objective
- Run gel on ligations from last night
- Redo/troubleshoot/transform based on gel + observations
Bench work
- Ran DNA gel (1% agarose):
- Lane 1: Ladder
- Lane 2: ----
- Lane 3: pTXB1 double digest 08/02 COULD NOT LOAD
- Lane 4: pTXB1 double digest; ligated with 'Old' buffer 08/03
- Lane 5: pTXB1 double digest; ligated with 'New' buffer 08/03
- Lane 6: ----
- Lane 7: pKK223-3 BamHI digest 08/03 COULD NOT LOAD
- Lane 8: pKK223-3 BamHI digest; ligated with 'Old' buffer 08/03
- Lane 9: pKK223-3 BamHI digest; ligated with 'New' buffer 08/03
- Lane 10: ----
- Lane 11: pMXB10 QuikChange +His 08/03
- Lane 12: pMXB10 QuikChange (-)control 08/03
- rest blank
- Based on an inability to load the digested, cleaned samples, these were re-cleaned with more extensive drying steps in order to remove supposed excess alcohol. The alcohol (2-propanol) would keep the gel load samples from resting in lanes and also inhibit ligase activity.
- Re-ligate the test ligation reactions done yesterday with re-cleaned plasmids.
- 'Overloaded' ligation of pTXB1.His and pTXB1 from 7/27 (50μL total volume)
- 20μL ddBSA insert + 10μL pTXB1 + 5μL ligase buffer ('New') + 13μL dH2O + 2μL T4 DNA ligase
- 20μL ddBSA insert + 10μL pTXB1.His + 5μL ligase buffer + 13μL dH2O + 2μL T4 DNA ligase
- 0μL insert + 10μL pTXB1 + 5μL ligase buffer + 33μL dH2O + 2μL T4 DNA ligase
- 0μL insert + 10μL pTXB1.His + 5μL ligase buffer + 33μL dH2O + 2μL T4 DNA ligase
Results
Gel
|