Molecular biology

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	==Reagents & Protocols==		==Reagents & Protocols==
-	*[[media:mba.pdf|Commonly used reagents and equipment in molecular biology (PDF)]]	+	===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)]]
-	*LIC cloning introduction [[media:LICnovagen.pdf|LIC introduction]] - Ligation Independent Cloning	+	**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
-	*LIC cloning protocol (Berkeley Structural Genomics Center)	+
-	**[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-rxn-transf.pdf LIC transformation]	+
	*Electrocompetent cell [[media:ElectrocompetentEcoli.pdf|Protocol (PDF)]]		*Electrocompetent cell [[media:ElectrocompetentEcoli.pdf|Protocol (PDF)]]
-	*[[Concentration of antibiotics commonly used]]	+	====LIC cloning====
-	==Softwares==	+	*Reference:
-	*[[NIH Image]]: Gel analysis software	+	<biblio>LIC pmid=2235490</biblio>
-	*[http://serialbasics.free.fr/Serial_Cloner.html Serial Cloner]: DNA sequence management (PCR, restriction maps, etc.)	+	*LIC cloning introduction [[media:LICnovagen.pdf|LIC introduction]] - Ligation Independent Cloning
-	*Bioedit	+	**LIC cloning protocol (Berkeley Structural Genomics Center)
-	*BioX (Mac OS X only)	+	***[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-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

Contents 1 Reagents & Protocols 1.1 Recipes 1.2 Protocols 1.2.1 LIC cloning 1.2.2 Conjugation 1.2.3 Gene replacement 2 Tools 2.1 webtools 2.2 softwares 3 Links

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

1. quality score
2. good sequencing result vs. bad sequencing result (see below example)

good vs. bad sequencing 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:

1. Error fetching PMID 2235490: [LIC]

• 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)

Conjugation

• Donor: E. coli S17-1

Gene replacement

• E. coli knockout by the Wanner method (Lambda Red Recombinase)

Tools

webtools

1. Oligo Tm calculation

softwares

1. NIH Image: Gel analysis software
2. Serial Cloner: DNA sequence management (PCR, restriction maps, etc.)
3. BioX (Mac OS X only)
4. Biological sequence editor Bioedit
5. Jmol

Links

• NCBE practical protocols: very good illustration for undergraduate students