Team:Wash U/Protocol
From 2009.igem.org
(Difference between revisions)
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:General Description | :General Description | ||
'''Materials''' | '''Materials''' | ||
- | *Spirulina Powder | + | * Spirulina Powder |
- | *dI water | + | * dI water |
- | *Methanol | + | * Methanol |
- | *HgCl2 | + | * HgCl2 |
- | *Trifluoroacetic acid (TFA) | + | * Trifluoroacetic acid (TFA) |
- | *Trichloroacetic acid (TCA) | + | * Trichloroacetic acid (TCA) |
- | *Acetonitrile | + | * Acetonitrile |
'''Procedures''' | '''Procedures''' | ||
#Rehydrate Spirulina powder in dI water (30ml/g dry weight) for 10 min. | #Rehydrate Spirulina powder in dI water (30ml/g dry weight) for 10 min. | ||
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:Description | :Description | ||
'''Materials''' | '''Materials''' | ||
- | *Centrifuge/screw top tubes | + | * Centrifuge/screw top tubes |
- | *50% glycerol sol | + | * 50% glycerol sol |
- | *LB Cultures | + | * LB Cultures |
'''Procedures''' | '''Procedures''' | ||
# Label centrifuge/screw top tubes | # Label centrifuge/screw top tubes | ||
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<font size="2"> | <font size="2"> | ||
'''Lysogeny Broth (LB) Media''' | '''Lysogeny Broth (LB) Media''' | ||
- | *10g Tryptone | + | * 10g Tryptone |
- | *5g Yeast Extract | + | * 5g Yeast Extract |
- | *10g NaCl | + | * 10g NaCl |
- | *15g Agar | + | * 15g Agar |
- | *up to 1 Liter H20 | + | * up to 1 Liter H20 |
- | *adjust pH to 7.5 | + | * adjust pH to 7.5 |
'''ATCC medium: Van Niel's yeast agar''' | '''ATCC medium: Van Niel's yeast agar''' | ||
- | *1.0 g K2HPO4 | + | * 1.0 g K2HPO4 |
- | *0.5 g MgSO4 | + | * 0.5 g MgSO4 |
- | *10.0 g yeast extract | + | * 10.0 g yeast extract |
- | *20.0 g agar | + | * 20.0 g agar |
- | *1.0 L tap water | + | * 1.0 L tap water |
- | *Adjust pH to 7.0-7.2 | + | * Adjust pH to 7.0-7.2 |
'''Super Optimal Broth (SOB)''' | '''Super Optimal Broth (SOB)''' | ||
- | *2% w/v bacto-tryptone (20g) | + | * 2% w/v bacto-tryptone (20g) |
- | *0.5% w/v bacto-yeast extract (5g) | + | * 0.5% w/v bacto-yeast extract (5g) |
- | *8.56mM NaCl (0.5g) or 10mM NaCl (0.584g) | + | * 8.56mM NaCl (0.5g) or 10mM NaCl (0.584g) |
- | *2.5mM KCl (0.186g) | + | * 2.5mM KCl (0.186g) |
- | *ddH2O to 1L | + | * ddH2O to 1L |
'''Super Optimal Broth with Catabolite repression (SOC)''' | '''Super Optimal Broth with Catabolite repression (SOC)''' | ||
- | *In addition to all of the SOB components, | + | * In addition to all of the SOB components, |
- | *10mM MgCl2 (0.952g) | + | * 10mM MgCl2 (0.952g) |
- | *20mM glucose (3.603g) | + | * 20mM glucose (3.603g) |
'''50X TAE Buffer''' | '''50X TAE Buffer''' | ||
- | *242 g Tris base (2-amino-2-hydroxymethyl-propane-1,3-diol) (= 2 mole) | + | * 242 g Tris base (2-amino-2-hydroxymethyl-propane-1,3-diol) (= 2 mole) |
- | *57.1 ml glacial acetic acid (= 100% acetic acid) (57.19 ml = 1 mole) | + | * 57.1 ml glacial acetic acid (= 100% acetic acid) (57.19 ml = 1 mole) |
- | *100 ml 0.5 M Na2 EDTA (pH 8.0) | + | * 100 ml 0.5 M Na2 EDTA (pH 8.0) |
- | *ddH2O to 1L | + | * ddH2O to 1L |
'''5X TBE Buffer''' | '''5X TBE Buffer''' | ||
- | *53 g of Tris base (CAS# 37186) | + | * 53 g of Tris base (CAS# 37186) |
* 27.5 g of boric acid (CAS# 11280) | * 27.5 g of boric acid (CAS# 11280) | ||
- | *20 ml of 0.5 M EDTA (CAS# 60004) (pH 8.0) | + | * 20 ml of 0.5 M EDTA (CAS# 60004) (pH 8.0) |
- | *ddH2O to 1L | + | * ddH2O to 1L |
'''Tris EDTA (TE) buffer 5X 1L''' | '''Tris EDTA (TE) buffer 5X 1L''' | ||
- | *750 mL d-H20 | + | * 750 mL d-H20 |
- | *242 g Tris base | + | * 242 g Tris base |
- | *57.1 mL glacial acetic acid | + | * 57.1 mL glacial acetic acid |
- | *100 mL 0.5M EDTA (93.05 g EDTA in 500 mL d-H20, pH ~8.0) | + | * 100 mL 0.5M EDTA (93.05 g EDTA in 500 mL d-H20, pH ~8.0) |
- | *fill to 1 L | + | * fill to 1 L |
- | *adjust pH to 8.5 | + | * adjust pH to 8.5 |
'''Agarose Gel (for electrophoresis of DNA >100bp)''' | '''Agarose Gel (for electrophoresis of DNA >100bp)''' | ||
- | *50 mL 1X TAE buffer | + | * 50 mL 1X TAE buffer |
- | *0.8 g agarose | + | * 0.8 g agarose |
- | *2.5 microliters EtBr (Caution: EtBr is a known carcinogen) | + | * 2.5 microliters EtBr (Caution: EtBr is a known carcinogen) |
- | *Note: Jacob suggested adding 1.0 microliter EtBr to gel and 3.0 microliters to TAE buffer in rig. | + | * Note: Jacob suggested adding 1.0 microliter EtBr to gel and 3.0 microliters to TAE buffer in rig. |
[[Team:Wash_U/Protocol#Procedures|Back To Top]] | [[Team:Wash_U/Protocol#Procedures|Back To Top]] | ||
{{WashUbottom}} | {{WashUbottom}} |
Revision as of 15:49, 9 July 2009