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Team:BYU Provo/Notebook/Cholera - Enzymes/Protocols
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Cholera-Enzyme Protocols
Salt LB recipe
To 950 ml distilled H2O add 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 98°C for 02:00
98°C for 00:30
65°C for 00:30
72°C for 04:00
X35
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 95 for 2 95 for .5 50 for .5 72 for 1 72 for 1
Any deviations from these protocols are listed in the notebook when they were used. |
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