Haynes:Protocols: Difference between revisions
| (6 intermediate revisions by the same user 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_Lipo | Transfection - Lipofectamine]] - Transfection of plasmid DNA into cells with Lipofectamine | |||
| Line 39: | Line 41: | ||
==Real Time Quantitative PCR== | ==Real Time Quantitative PCR== | ||
* [[Haynes:UPLassay | Universal Probe Library (UPL) assay]] | * [[Haynes:UPLassay | Universal Probe Library (UPL) assay]] - for the Roche Light Cycler 480 | ||
==Resources | ==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 58: | Line 64: | ||
<div style="width: 350px"> | <div style="width: 350px"> | ||
'''Bromo-Blue/X-cyanol Loading Buffer, | '''Bromo-Blue/X-cyanol Loading Buffer, 20X''' {{hide| | ||
Formula: [ | Formula: [60% glycerol; 12.5 mg/mL bromophenol blue; 12.5 mg/mL xylene cyanol]<br> | ||
Volume: 20 mL | Volume: 20 mL | ||
* | * 100% glycerol, 12 mL | ||
* Bromophenol blue, 250 mg | * Bromophenol blue, 250 mg | ||
* Xylene cyanol, 250 mg | * Xylene cyanol, 250 mg | ||
Start with 18 mL dH<sub>2</sub>O. Add in glycerol 6 mL at a time and mix by pipetting up and down several times (it is very viscous). Add dyes. | |||
}} | }} | ||
| Line 79: | Line 85: | ||
Bring up to total volume with dH<sub>2</sub>O. Store at 4°C. Add 100x protease inhibitor cocktail* (1:100) immediately before use. | Bring up to total volume with dH<sub>2</sub>O. Store at 4°C. Add 100x protease inhibitor cocktail* (1:100) immediately before use. | ||
}} | |||
'''DNA Ladder mix, Gene Ruler 1 kb Plus''' {{hide| | |||
Formula: [0.5 ng/ 10 μL Fermentas Gene Ruler 1 kb Plus; 1x loading dye]<br> | |||
Volume: 1000 μL | |||
* Gene Ruler plus (0.5 ng/μL), 100 μL | |||
* 20x loading dye, 50 μL | |||
* dH<sub>2</sub>O, 850 μL | |||
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. | |||
}} | }} | ||
Revision as of 23:37, 8 January 2013
Protocols
DNA Assembly
- BioBrick Parts Assembly: an Overview
- Gibson Assembly
- Type IIS Assembly - similar to Golden Gate, Golden Braid, and Moclo
Cell Culture: Bacteria
- Chemically competent cell prep
- Glycerol Stocks - for long term -80°C storage
- Transformation of E. coli with plasmid DNA
Cell Culture: Mammalian
- Media formulas - cell line-specific formulas
- Transfection - Lipofectamine - Transfection of plasmid DNA into cells with Lipofectamine
Protein
- Bradford assay - measure protein concentration
Real Time Quantitative PCR
- Universal Probe Library (UPL) assay - for the Roche Light Cycler 480
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 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
- Appropriate incomplete medium, 500 mL
- Fetal bovine serum (FBS), 50 mL
- Penicillin/ streptomycin (pen-strep), 5 mL
- Appropriate antibiotics, depending upon formula
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.
