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Team:BYU Provo/Notebook/Cholera - Enzymes/Protocols
From 2013.igem.org
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Cholera-Enzyme Protocols
Salt LB recipe
To 950 ml distilled H2O add
Adjust pH to 7.8
Fill to 1000 ml and autoclave
Phusion PCR (50ul reaction)
For each sample add:
Set the PCR machine to run the following cycle with the middle three phases being repeated 35 times
Hold at 4°C
Taq polymerase PCR (50ul reaction)
For each sample add:
Set the PCR machine to run the following cycle with the middle three phases being repeated 35 times
Hold at 4°C
Restriction Digest (50ul reaction)
For each sample add:
Place in 37°C incubator or water bath for 1.5 hours
Ligation
For each ligation reaction add
Incubate the reaction at room temperature for 30 minutes
Transformations
Set two heat blocks at 42°C and 37°C respectively. Thaw 25 ul of chemically competent cells on ice and add 5 ul of DNA to be cloned in. Place on ice for approximately 5 minutes. Heat shock at 42°C for 1 minute and immediately place back on ice for 2-5 minutes. Add 500 ul LB to the reaction and incubate at 37°C for 30 minutes. Plate 100 ul of cell on appropriate antibiotic plate and incubate at 37°C overnight.
V. cholerae Biofilm Degradation Assay
For each sample add 1 ml salt LB and 50 ul overnight seed. Add appropriate
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