DNA转化

 

DNA 转化

 

Chemical Transformation

・         Transformation of Competent Cells (RbCl2 Method) (Goldberg Lab)
Very nice protocol for E. Coli transformation including competent cell preparation and recipes

・         Transformation of E. coli ( Crawford Lab)

・         Standard Transformation Protocol ( Promega)
You can find general and useful protocol for the whole experiment of plasmid cloning from DNA and vector preparation, ligation, transformation, plasimd preparation and transfection...

・         Transformation  of E. Coli ( Life Technologies)
How to stabilize unstable inserts and increasing plasmid yields

・         Transformation of E.Coli, M13 Cloning   ( Life Technologies)

・         Five-Minute Transformation (Chun-Ming Liu)
Quick E. Coli transformation method. It works! 

・         Transformation  (Lazo Lab) 
One-step PEG method

・         Protocol for Transforming Competent E. coli Cells with Plasmid DNA (Dr. Chastain)
Very basic method for students.

• Transformation of Aspergillus niger (Molecular Genetics Network Lab)
• Transformation of Aspergillus nidulans (Molecular Genetics Network Lab)
     
• Large-scale transformation procedures (YGAC)
  This protocol merges a typical LiAc protocol with the reducing agent component of the One-step method. It gives >10³ / ug efficiency for our strain. Recipe is for 800 transformations (90 mls cells).

Electroporation

・         Transformation of E. coli by Electroporation ( Julie B. Wolf, UMBC)
provides details for preparing cell and DNA and electroporation procedure

・         Transformation of E. Coli by Electroporation (Hoshi Lab)

・         Electroporation of P. Aeruginosa (Goldberg Lab)
Detailed protocol for preparing electroporation cell and electroporation procedure.

・         Transformation of E. Coli by Electroporation (Goldberg Lab)
Very nice protocol for electroporation including cell preparation and recipes  

・         Transformation of E. Coli by Electroporation (Crawford Lab)

・         Preparation of DNA for Electroporation (Life Technologies)

Preparation of Competent Cell

• Preparation of Competent Cells (Donis Keller Lab)
  Detailed method for competent cell preparation
     
• Preparation of Competent Cell (Ubidium Chloride Method ) (NWFSC)
    

・         Preparation of Competent Cell (Inoue "ultra-competent" method ) (NWFSC)

・         Preparation of Competent Cell (PDF)(Templenton Lab)
A great method for making highly competent cells, simply.

・         Preparation of Competent E. coli Cells (Waters Lab)

・         Preparation of Competent Cell ( Crawford Lab)

・         One-step competent E. coli cells (Dept. of Clinical Pharmacology, University of Berne, Switzerland)
A quick method of preparing competent cell. Transformation efficiency by this protocol is 10⁶ to 10⁷ cfu/ m g.

・         Preparing and Using Bacterial Cells for Electroporation (PMCI Research)
Detailed protocol for cell preparation and electroporation

・         Electroporation Competent E.Coli Preparation (Hoshi Lab)

・         Electroporation Competent E.Coli Preparation ( Bowtell Lab)
Including procedures for cell preparation and electroporation

Clone Screening and others

・         Transfer of DNA from Bacterial Colonies to Nylon Membrane or Nitrocellulose (Donis Keller Lab)
To transfer DNA from bacterial colonies to nylon (or nitrocellulose) membrane so that a library can be screened by hybridization.

・         Screening clone by PCR ( Herskowitz Lab)
Add colony directly into PCR tubes without plasmid preparation

• Bacterial Colony PCR (Karl Clark)
  Allows quick identification of correct clones if primers exist that allow clone identification by size or hybridization.
    

・         Colony lifts
Lift plasmid colonies onto nitrocellulose filter for screen

・         Toothpick Plasmid Assay (Donis Keller Lab)
The toothpick assay is used to identify bacterial colonies containing recombinant plasmids following transformation and to obtain an approximation of the plasmid insert sizes.

・         Making Glycerol Stocks of Transformed Cells (Life Technologies)
For long-term storage of transformed cell

・         Preparing Lawn Cells for M13 Cloning (LTI)
Lawn cells require the F' episome for M13 infection and may be prepared using...

・         PCR Screening of Transformants ( Crawford Lab)
Quick screening of transformants without minipreps

・         Calculating Transformation Efficiency (LTI)

・         Bacteria Storage (Gerard R. Lazo)
Preserve E. coli strains in stab agar or glycerol for mid-term storage and lyophilize for long-term storage.

・         In situ screening of bacterial colonies (NUNC)
Positive clones are selected after plasmid cloning. This is often done by DNA probe hybridization in situ to plasmid DNA containing E.coli cells. The following protocol can be used for most plasmid cloning events in E.coli cells.

・         Slot Lysis (Hancock Lab) (Accessible only by IE)
This is a quick method to determine the presence of cloned fragments in plasmids after ligation/transformation.

・         Media and buffers for Aspergillus transformation (Molecular Genetics Network Lab)

Transformation Trouble Shooting 

・         Trouble Shooting of E Coli Transformation (LTI)
Very useful trouble shooting guide for common transformation problems like no colonies, all white or blue colonies, restriction resistance of your plasmid and more. If you are new to transformation, it's better to have a look.