Team:BYU Provo/Notebook/Cholera - Enzymes/Protocols

Cholera Enzyme

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Protocols

• 24.00 g NaCl
• 11.90 g MgCl2-6H2O
• 02.00 g CaCl2-2H2O
• 00.85 g KCl

Adjust pH to 7.8
Add

• 10.00 g Bacto-tryptone
• 05.00 g Yeast extract

Fill to 1000 ml and autoclave

V. cholerae Biofilm growth

Start a 5 ml overnight of V. cholerae in SSW LB and incubate at 30° for 72 hours. In a separate culture tube add 4 ml SSW LB and 50 ul overnight culture. Incubate at 30°C for at least 48 hours.

Phusion PCR (50ul reaction)

For each sample add:

• 35 ul ddH2O
• 10 ul 5X Phusion buffer
• 1.5 ul 10mM dNTP's
• 1 ul each Primer (50 uM stock)
• 1 ul appropriate diluted template DNA
• 0.5 ul Phusion Polymerase

Set the PCR machine to run the following cycle with the middle three phases being repeated 35 times

• 98°C for 02:00
• 98°C for 00:30
• 65°C for 00:30
• 72°C for 04:00
• 72°C for 8:00

Hold at 4°C

Taq polymerase PCR (50ul reaction)

For each sample add:

• 40 ul ddH2O
• 5 ul 10X TAQ buffer
• 1.5 ul 10 mM dNTP's
• 1 ul each Primer (50 uM stock)
• 1 ul appropriate diluted template DNA
• 0.5 ul TAQ Polymerase

Set the PCR machine to run the following cycle with the middle three phases being repeated 35 times

• 95 for 2:00
• 95 for 0:30
• 50 for 0:30
• 72 for 1:00
• 72 for 1:00

Hold at 4°C

Restriction Digest (50ul reaction)

For each sample add:

• 14 ul ddH2O
• 5 ul 10X NEB buffer
• 0.5 ul 100X BSA
• 30 ul DNA sample
• 2 ul each restriction enzyme

Place in 37°C incubator or water bath for 1.5 hours

Ligation

For each ligation reaction add

• 6.5 ul H2O
• 1.5 ul 10X ligase buffer
• 1 ul T4 DNA ligase
• 3 ul vector
• 3 ul insert

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

• Revised from original Biofilm Degradation Assay from Pitts, B.; Hamilton, M.; Zelver, N.; Stewart, P. A Microtiter-Plate Screening Method for Biofilm Disinfection and Removal. J. Micro Methods 2003, 54, 269-276. V. cholerae biofilms are more apt to free float rather than develop on solid surfaces.

For each sample add 1 ml SSW LB and 50 ul overnight seed. Add appropriate amount of degrading enzymes to each sample labeled time = 0 hours. Incubate at 30°C for 48.