Team:Caltech/Recipes
From 2011.igem.org
(Difference between revisions)
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<p>'''Gibson Mix (1.33x)'''</p> | <p>'''Gibson Mix (1.33x)'''</p> | ||
+ | For 25 aliquots of 15 μl each: | ||
* 50 μl of Taq Ligase | * 50 μl of Taq Ligase | ||
* 100 μl of 5x isothermal buffer | * 100 μl of 5x isothermal buffer | ||
Line 48: | Line 49: | ||
* 216.75 μl of Nuclease-free water | * 216.75 μl of Nuclease-free water | ||
* (375 μl total) | * (375 μl total) | ||
- | + | <br/> | |
- | + | ||
- | + | ||
<p>'''Phusion PCR'''</p> | <p>'''Phusion PCR'''</p> | ||
+ | For a 50uL reaction: | ||
* 1 uL template DNA | * 1 uL template DNA | ||
* 2..5 uL fwd and rev primer | * 2..5 uL fwd and rev primer | ||
Line 68: | Line 68: | ||
*adjust pH to 7.5 using 1M NaOH | *adjust pH to 7.5 using 1M NaOH | ||
*autoclave | *autoclave | ||
- | *after cooling below 50 | + | *after cooling below 50°C, add 5 mL filter-sterilized 20% glucose solution.<br/><br/> |
+ | |||
+ | <p>'''Taq PCR (for 16s insert)'''</p> | ||
+ | For a 25uL reaction: | ||
+ | *sterile water: 19.8uL | ||
+ | *taq buffer (10x): 2.5uL | ||
+ | *dNTP (10mM): 0.6uL | ||
+ | *Fwd primer (10uM): 0.5uL | ||
+ | *Rev primer (10uM): 0.5uL | ||
+ | *template DNA : 1uL | ||
+ | *Taq (5U/ul): 0.1uL | ||
<br/>}} | <br/>}} |
Revision as of 21:59, 27 July 2011
Project |
Back to Timeline . Back to Methods 50% glycerol Stock:
Agar/LB plate (Autoclaved):
Ampicillin Stock
Chloramphenicol Stock
Enrichment Minimal Media
Gibson Mix (1.33x) For 25 aliquots of 15 μl each:
Phusion PCR For a 50uL reaction:
SOC Media for 250 ml (adapted from [http://openwetware.org/wiki/SOC OpenWetWare])
Taq PCR (for 16s insert) For a 25uL reaction:
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