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Team:BYU Provo/Notebook/Cholera - Enzymes/Protocols
From 2013.igem.org
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To 950 ml distilled H2O add | 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 | + | Adjust pH to 7.8 <br> |
Add | Add | ||
| - | + | *10.00 g Bacto-tryptone | |
| - | + | *05.00 g Yeast extract | |
Fill to 1000 ml and autoclave | Fill to 1000 ml and autoclave | ||
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For each sample add: | 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 | 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 | X35 | ||
| - | + | *72°C for 8:00 | |
Hold at 4°C | Hold at 4°C | ||
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For each sample add: | 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 2 | ||
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<br> | <br> | ||
| - | Any deviations from these protocols are listed in the notebook when they were used | + | ===Any deviations from these protocols are listed in the notebook when they were used=== |
Revision as of 21:37, 27 September 2013
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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
X35
Hold at 4°C
Taq polymerase PCR (50ul reaction)
For each sample add:
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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