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

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

PCR Product 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