Molecular biology
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- | *[[media:mba.pdf|Commonly used reagents and equipment in molecular biology (PDF)]] | + | {| align=right cellpadding=15 |
+ | |__TOC__ | ||
+ | |} | ||
+ | ==Reagents & Protocols== | ||
+ | ===Recipes=== | ||
+ | ;*[[Chemicals]] | ||
+ | ;*[[media:mba.pdf|Commonly used reagents and equipment in molecular biology (PDF)]] | ||
+ | ;*[[Concentration of antibiotics commonly used]] | ||
+ | ;*For DNA extraction (to remove some protein contaminants & inactivate enzymes): Phenol:Chloroform=1:1(v:v) solution | ||
+ | ;*DNA sequencing check list | ||
+ | :#[http://en.wikipedia.org/wiki/Phred_quality_score quality score] | ||
+ | :#good sequencing result vs. bad sequencing result (see below example) | ||
+ | ::[[image:sequencingresult.jpg|center|thumb|good vs. bad sequencing example]] | ||
+ | |||
+ | ===Protocols=== | ||
*[[PCR primer|PCR]] - General rules for primer design | *[[PCR primer|PCR]] - General rules for primer design | ||
*[[Genomic DNA extraction]] | *[[Genomic DNA extraction]] | ||
*Competent cell preparation [[media:competentcell.pdf|BSGC protocol (PDF)]] | *Competent cell preparation [[media:competentcell.pdf|BSGC protocol (PDF)]] | ||
+ | **Easy & rapid competent cell preparation [http://www.personal.psu.edu/faculty/d/s/dsg11/labmanual/DNA_manipulations/Comp_bact_by_TSS.htm reference link] - by HJKo | ||
+ | *Electrocompetent cell [[media:ElectrocompetentEcoli.pdf|Protocol (PDF)]] | ||
+ | ====LIC cloning==== | ||
+ | *Reference: | ||
+ | <biblio>LIC pmid=2235490</biblio> | ||
*LIC cloning introduction [[media:LICnovagen.pdf|LIC introduction]] - Ligation Independent Cloning | *LIC cloning introduction [[media:LICnovagen.pdf|LIC introduction]] - Ligation Independent Cloning | ||
- | *LIC cloning protocol (Berkeley Structural Genomics Center) | + | **LIC cloning protocol (Berkeley Structural Genomics Center) |
- | **[http://www.strgen.org/protocols/lic-vector-prep.pdf Vector preparation] | + | ***[http://www.strgen.org/protocols/lic-vector-prep.pdf Vector preparation] |
- | **[http://www.strgen.org/protocols/lic-insert-prep.pdf Insert preparation] | + | ***[http://www.strgen.org/protocols/lic-insert-prep.pdf Insert preparation] |
- | **[http://www.strgen.org/protocols/lic-rxn-transf.pdf LIC transformation] | + | ***[http://www.strgen.org/protocols/lic-rxn-transf.pdf LIC transformation]: |
- | * | + | ****mix insert and vector in appropriate amounts |
+ | ****incubate 5~30 min(20 min) at room temperature | ||
+ | ****do transformation in E. coli (recommend to use electro-competent cells) | ||
+ | |||
+ | ====Conjugation==== | ||
+ | *Donor: <i>E. coli</i> [http://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=47055&Template=hosts S17-1] | ||
+ | ====Gene replacement==== | ||
+ | *[[E. coli knockout]] by the Wanner method (Lambda Red Recombinase) | ||
+ | |||
+ | ==Tools== | ||
+ | ===webtools=== | ||
+ | #[http://www.basic.northwestern.edu/biotools/oligocalc.html Oligo Tm calculation] | ||
+ | ===softwares=== | ||
+ | #[[NIH Image]]: Gel analysis software | ||
+ | #[http://serialbasics.free.fr/Serial_Cloner.html Serial Cloner]: DNA sequence management (PCR, restriction maps, etc.) | ||
+ | #[http://www.ebioinformatics.org/ BioX] (Mac OS X only) | ||
+ | #Biological sequence editor [http://www.mbio.ncsu.edu/BioEdit/bioedit.html Bioedit] | ||
+ | #[http://en.wikipedia.org/wiki/Jmol Jmol] | ||
+ | ==Links== | ||
+ | *[http://www.ncbe.reading.ac.uk/ncbe/protocols/ NCBE practical protocols]: very good illustration for undergraduate students |
Latest revision as of 07:30, 16 January 2013
|
Reagents & Protocols
Recipes
- Chemicals
- Commonly used reagents and equipment in molecular biology (PDF)
- Concentration of antibiotics commonly used
- For DNA extraction (to remove some protein contaminants & inactivate enzymes): Phenol:Chloroform=1:1(v:v) solution
- DNA sequencing check list
- quality score
- good sequencing result vs. bad sequencing result (see below example)
Protocols
- PCR - General rules for primer design
- Genomic DNA extraction
- Competent cell preparation BSGC protocol (PDF)
- Easy & rapid competent cell preparation reference link - by HJKo
- Electrocompetent cell Protocol (PDF)
LIC cloning
- Reference:
Error fetching PMID 2235490:
- Error fetching PMID 2235490:
- LIC cloning introduction LIC introduction - Ligation Independent Cloning
- LIC cloning protocol (Berkeley Structural Genomics Center)
- Vector preparation
- Insert preparation
- LIC transformation:
- mix insert and vector in appropriate amounts
- incubate 5~30 min(20 min) at room temperature
- do transformation in E. coli (recommend to use electro-competent cells)
- LIC cloning protocol (Berkeley Structural Genomics Center)
Conjugation
- Donor: E. coli S17-1
Gene replacement
- E. coli knockout by the Wanner method (Lambda Red Recombinase)
Tools
webtools
softwares
- NIH Image: Gel analysis software
- Serial Cloner: DNA sequence management (PCR, restriction maps, etc.)
- BioX (Mac OS X only)
- Biological sequence editor Bioedit
- Jmol
Links
- NCBE practical protocols: very good illustration for undergraduate students