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| | '''Purification of DNA''' | | '''Purification of DNA''' |
| − | Golden Gate Assembly Protocol for BsmB1
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| − | GGA mixture contains:
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| − | 1 µL (50 ng) Plasmid
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| − | 1 µL Promoter
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| − | 1 µL 10X Promega Ligase Buffer
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| − | 6 µL dH2O
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| − | 0.5 µL BsmB1 high fidelity (HF) restriction enzyme
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| − | 9.5 µL volume
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| − | Place tubes into 55°C water bath for 15 minutes.
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| − | Add 0.5 µL T4 DNA Ligase to the mixture.
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| − | Turn on the PCR machine. Put tube into machine.
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| − | Program it for the following cycles:
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| − | • 20 cycles
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| − | • 55°C for 10 minutes
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| − | • 37°C for 1 minute
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| − | • 16°C for 1 minute
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| − | • 22°C holding temperature
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| − | This DNA ligation is ready for transformation.
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| − | You can increase the number of cycles to 30 if you want to increase yield. However, we have gotten good success with as few as 5 cycles.
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| − | Transformation of GGA BsmB1
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| − | For each reaction done you will need an agar plate and 50µL of competent cells mixed with competent cell buffer. Mix the 50µL of cell mixture with the GGA product. Place the entire content of the tube on the plate, spread and incubate.
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| | [[Isolation of Genomic DNA from Bacteria]] | | [[Isolation of Genomic DNA from Bacteria]] |
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Revision as of 21:44, 4 March 2013
Purification of DNA
Isolation of Genomic DNA from Bacteria
PCR
Standard PCR
Resuspending Oligos
Colony PCR
Recombinant DNA Production
Pouring an Agarose Gel
BioBrick Digestions for Fragment and Vector Preparation
Fragment Purification
Gibson Assembly
Direct Synthesis with Overlapping Oligos
Annealing Oligos for Cloning
Ethanol Precipitation of Vector DNA
Reducing Background from Double Digested Vector
Ligation and Transformation
BioBrick Ligations
Ligation and Transformation
Bacterial Media
Screening Clones
Diagnostic RP Digestion for Checking Insert Size
DNA Sequencing at Iowa State University
What to do with a new clone
Measuring Phenotypes
Measuring Fluorescence in Bacteria
