Haynes:Protocols

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Recipes)
(Recipes)
(5 intermediate revisions not shown.)
Line 20: Line 20:
** [[Haynes:Assembly101 | Model Procedure for Assembling BioBrick Parts: Classic Ligation for Beginners]]
** [[Haynes:Assembly101 | Model Procedure for Assembling BioBrick Parts: Classic Ligation for Beginners]]
* [[Haynes:Gibson_Assembly | Gibson Assembly]]
* [[Haynes:Gibson_Assembly | Gibson Assembly]]
 +
* [[Haynes:TypeIIS_Assembly | Type IIS Assembly]] - similar to Golden Gate, Golden Braid, and Moclo
<br>
<br>
Line 32: Line 33:
==Cell Culture: Mammalian==
==Cell Culture: Mammalian==
* [[Haynes:MamCultureMedia | Media formulas]] - cell line-specific formulas
* [[Haynes:MamCultureMedia | Media formulas]] - cell line-specific formulas
-
* [[Haynes:TransfectionPlasmid | Transfection]] - Transfection of plasmid DNA into cells
+
* [[Haynes:TransfectionPlasmid_Lipo | Transfection - Lipofectamine]] - Transfection of plasmid DNA into cells with Lipofectamine
 +
 
==Protein==
==Protein==
Line 39: Line 41:
==Real Time Quantitative PCR==
==Real Time Quantitative PCR==
-
* [[Haynes:UPLassay | Universal Probe Library (UPL) assay]]: for the Roche Light Cycler 480
+
* [[Haynes:UPLassay | Universal Probe Library (UPL) assay]] - for the Roche Light Cycler 480
-
==Resources at OpenWetWare==
+
==RNA & cDNA protocols==
 +
* [[Haynes:TRIzol_RNeasy | RNA mini prep - TRIzol/ RNeasy column]]
 +
 
 +
 
 +
==Other Resources - OpenWetWare==
* [http://openwetware.org/wiki/E._coli_genotypes E. coli Strains]  
* [http://openwetware.org/wiki/E._coli_genotypes E. coli Strains]  
* [http://openwetware.org/wiki/Materials Materials]: buffer formulas; specific information on commonly used reagents (''e.g.'', LB broth, ampicillin, restriction enzymes, etc.)
* [http://openwetware.org/wiki/Materials Materials]: buffer formulas; specific information on commonly used reagents (''e.g.'', LB broth, ampicillin, restriction enzymes, etc.)
Line 82: Line 88:
'''DNA Ladder mix, Gene Ruler 1 kb Plus''' {{hide|  
'''DNA Ladder mix, Gene Ruler 1 kb Plus''' {{hide|  
-
Formula: [0.5 ng/ 10 μL Fermentas Gene Ruler 1 kb Plus]<br>
+
Formula: [0.5 ng/ 10 μL Fermentas Gene Ruler 1 kb Plus; 1x loading dye]<br>
Volume: 1000 μL
Volume: 1000 μL
* Gene Ruler plus (0.5 ng/μL), 100 μL
* Gene Ruler plus (0.5 ng/μL), 100 μL
Line 89: Line 95:
Add dH<sub>2</sub>O directly to the supplier's tube (containing 100 μL of ladder). Add the 20x loading dye. Mix. Aliquot 200 μL portions into five 1.5 mL tubes. Store at -20°C.
Add dH<sub>2</sub>O directly to the supplier's tube (containing 100 μL of ladder). Add the 20x loading dye. Mix. Aliquot 200 μL portions into five 1.5 mL tubes. Store at -20°C.
 +
}}
 +
 +
'''LB (Lennox) Agar plates''' {{hide|
 +
Volume: 1 L
 +
* Bacto-agar, 15 g
 +
* Caesin tryptone, 10 g
 +
* Yeast extract, 5 g
 +
* NaCl, 5 g
 +
* 1 N NaOH, 1 mL
 +
 +
Dissolve in 1000 mL dH<sub>2</sub>O. Autoclave to sterilize. Cool to ~50°C (warm to touch) before adding antibiotics. Add antibiotic(s) and pour into sterile petri dishes. 1L should yield about 40 plates. Shortcut: Use 25 g Acros LB Broth, Lennox (BP9722-500) granules instead of caesin tryptone,  yeast extract, and NaCl.
}}
}}

Revision as of 22:15, 22 February 2013

link=Haynes_Lab
link=http://engineering.asu.edu/

   Home     Research     Publications     People     Contact     Protocols     Lab Resources     Personnel Info

Contents

Protocols

DNA Assembly


Cell Culture: Bacteria


Cell Culture: Mammalian


Protein


Real Time Quantitative PCR


RNA & cDNA protocols


Other Resources - OpenWetWare

  • E. coli Strains
  • Materials: buffer formulas; specific information on commonly used reagents (e.g., LB broth, ampicillin, restriction enzymes, etc.)


Software Guides



Recipes

Click "show" to expand each recipe, and "hide" to, well, hide it.

Bromo-Blue/X-cyanol Loading Buffer, 20X

Formula: [60% glycerol; 12.5 mg/mL bromophenol blue; 12.5 mg/mL xylene cyanol]
Volume: 20 mL

  • 100% glycerol, 12 mL
  • Bromophenol blue, 250 mg
  • Xylene cyanol, 250 mg

Start with 18 mL dH2O. Add in glycerol 6 mL at a time and mix by pipetting up and down several times (it is very viscous). Add dyes.

Chromatin Prep Buffer A

Formula: [10 mM HEPES (pH 7.9); 10 mM KCl, 1.5 mM MgCl2; 0.34 M sucrose; 10% glycerol; 1 mM dithiothreitol; 1x protease inhibitor cocktail]
Volume: 100 mL

  • 1 M HEPES pH 7.9, 1 mL
  • 1 M KCl, 1 mL
  • 1 M MgCl2, 150 μL
  • Sucrose, 11.6 g
  • 50% Glycerol, 20 mL
  • 1 M Dithiotreitol (DTT), 100 μL

Bring up to total volume with dH2O. Store at 4°C. Add 100x protease inhibitor cocktail* (1:100) immediately before use.

DNA Ladder mix, Gene Ruler 1 kb Plus

Formula: [0.5 ng/ 10 μL Fermentas Gene Ruler 1 kb Plus; 1x loading dye]
Volume: 1000 μL

  • Gene Ruler plus (0.5 ng/μL), 100 μL
  • 20x loading dye, 50 μL
  • dH2O, 850 μL

Add dH2O directly to the supplier's tube (containing 100 μL of ladder). Add the 20x loading dye. Mix. Aliquot 200 μL portions into five 1.5 mL tubes. Store at -20°C.

LB (Lennox) Agar plates

Volume: 1 L

  • Bacto-agar, 15 g
  • Caesin tryptone, 10 g
  • Yeast extract, 5 g
  • NaCl, 5 g
  • 1 N NaOH, 1 mL

Dissolve in 1000 mL dH2O. Autoclave to sterilize. Cool to ~50°C (warm to touch) before adding antibiotics. Add antibiotic(s) and pour into sterile petri dishes. 1L should yield about 40 plates. Shortcut: Use 25 g Acros LB Broth, Lennox (BP9722-500) granules instead of caesin tryptone, yeast extract, and NaCl.

LB Broth (Lennox)

Volume: 1 L

  • Caesin tryptone, 10 g
  • Yeast extract, 5 g
  • NaCl, 5 g
  • 1 N NaOH, 1 mL

Dissolve in 1000 mL dH2O. Autoclave to sterilize. Cool to room temperature before adding antibiotics. Shortcut: Use 25 g Acros LB Broth, Lennox (BP9722-500) granules instead of caesin tryptone, yeast extract, and NaCl.

Mammalian Cell Media

Volume: 500 mL

Add FBS and pen-strep directly to the incomplete medium in the original stock bottle. Cap and invert to mix. Filter sterilize using a vacuum filtration unit.


Personal tools