Molecular biology
From CSBLwiki
(Difference between revisions)
(→protocols) |
(→protocols) |
||
| Line 13: | Line 13: | ||
[[image:sequencingresult.jpg|left|thumb|good vs. bad sequencing example]] | [[image:sequencingresult.jpg|left|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]] | ||
| Line 30: | Line 30: | ||
****incubate 5~30 min(20 min) at room temperature | ****incubate 5~30 min(20 min) at room temperature | ||
****do transformation in E. coli (recommend to use electro-competent cells) | ****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) | *[[E. coli knockout]] by the Wanner method (Lambda Red Recombinase) | ||
Revision as of 13:13, 23 November 2010
|
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
