Objective
Continue trying to get BSA-intein construct cloned.
Bench work
- Ligation
- Using new digested DNA
- Previous purification could have been problematic, but hopefully the yield is higher and there is less ethanol this time.
- Insert: gel-purified NheI/SapI digested BSA PCR from yesterday
- Vector(His): gel-purified NheI/SapI/Phosphatased pTXB1His1 from yesterday
- Vector: gel-purified NheI/SapI/Phosphatased pTXB1 from yesterday
- 8.5μL Insert + 8.5μL Vector(His) + 2μL buffer + 1μL T4 DNA Ligase
- 8.5μL Insert + 8.5μL Vector + 2μL buffer + 1μL T4 DNA Ligase
- 8.5μL Vector(His) + 8.5μL H2O + 2μL buffer + 1μL T4 DNA Ligase (negative control)
- 8.5μL Vector + 8.5μL H2O + 2μL buffer + 1μL T4 DNA Ligase (negative control)
- → 16°C O/N
- → store @ -20°C
- Transformation
- 100μL DH10B + 5μL V(His)+I from yesterday
- 100μL DH10B + 5μL V+I from yesterday
- 100μL DH10B + 5μL V(His) neg ctrl from yesterday
- 100μL DH10B + 5μL V neg ctrl from yesterday
- heat shock time is 45s
- plate 100μL each
- Re-plate yesterday's NovaBlue transformation
- 450μL of each re-plated
- → 37°C O/N
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