DNA的酶学操作

DNA的酶学操作

• DNA Modifying Enzymes (Michael Blaber)
  Introduction to bacterial restriction/modification system. It provides very useful background knowledge about restriction digestion
     
• (Michael Blaber)
  Very useful guide to DNA modifying enzymes and their applications including methylases, polymerases, nucleases, ligases, kinase, phosphatase

Digestion

• Alphabetic List of Commercial Restriction Endonucleases and Their Isoschizomers (Fermentas)
    
• (Fermentas)
  • Enzymes Cleaving DNA on Both Sides of Their Recognition Sequence
       

·         (LaboratoryExperiments.com)
Basic procedures for high school students, But very useful if you are new to molecular biology.

·         Reaction (Crawford Lab)  

·         (Lazo Lab)
tips on restriction digestion

• (Donis Keller Lab)
  Tips for digestion
    
• Restriction Digestions (Dr. Chastain)
  There are generally 2 kinds of restriction digests: a diagnostic digest and a preparative digest
    
• (Donis Keller Lab)
  This is a generalized example of a restriction digest. Estimate the amount of DNA needed in your digest and scale up accordingly. To visualize a digest on an ethidium bromide-stained agarose gel, you will need to take the size of the fragments and the total size of the clone DNA into account (e. g. 10-50 ng of intact lambda-sized genomes (~50 kb) are easily seen on gels but if cut into small (~1kb fragments), the relative proportion of the clone DNA in each fragment is ~ 1/50 and more DNA (500-1000 ng) should be loaded in order to see them).
    
• (Donis Keller Lab)
  For digestion of human genomic DNA
     
• (Donis Keller Lab)
  To prepare intact mammalian chromosomal DNA for use in pulsed-field electrophoresis mapping experiments using rare cutting restriction enzymes.
   
• (Donis Keller Lab)
  To prepare high molecular weight DNA in a medium with high surface area, to facilitate complete digestion with restriction enzymes.The DNA prepared in agarose beads is also suitable for use in constructing YAC libraries.
     
• Preparation of Bacteriophage Lambda  Concatamers for use as Size Standards in Pulsed-Field Gel Electrophoresis (Donis Keller Lab)
  To produce molecular length size standards accurate in the range of 50-1000 kb.
      

·         (Julie B. Wolf, UMBC)

·         (Goldberg Lab)

·         (Hoshi Lab)
Detailed guide for performing digestion, stopping digestion

·         (Roe, OU)

·         Restriction Digestion in the Presence of Agarose (FMC)

·         Isolating DNA from the Restriction Digest Reaction Mixture (Hoshi Lab)

·         Restriction Enzyme Buffer Recipes
Prepare your customized buffer for a wide usr/localiety of restriction enzymes 

Modification

·         Making blunt ended DNA fragments with T4 DNA polymerase  (Molecular Genetics RecLab)

·         (Molecular Genetics RecLab)

·         (Molecular Genetics RecLab)

·         (Schneitz Lab)

·         (Vesicle Trafficking)

·         Bisulfite Modification (TTO)
Modify DNA with bisulfite for methylation analysis

·         Digestion of PCR Products (Fermentas)
Some basic facts you need to know to cut your PCR products.

·         Cleavage Efficiency Close to the Termini of PCR Fragments (Fermentas)
When designing PCR experiments in which the synthesized DNA fragment is to be subsequently digested with a RE, it is very important to determine how many extra nucleotides should be added to the 5’-end of the PCR primer next to the introduced recognition site to ensure efficient cleavage with the appropriate restriction endonuclease. Some restriction endonucleases cleave DNA poorly when their recognition sites are located at the ends of DNA fragments. Information on restriction endonuclease performance close to the end of DNA fragments may be helpful when planning the introduction of cleavage sites at DNA termini in PCR experiments.

Ligation

·         (Roe, OU)
DNA ligations are performed by incubating DNA fragments with appropriately linear zed cloning vector in the presence of buffer, rATP, and T4 DNA ligase.

·         Standard Ligation (Schneitz Lab)
Detailed guide for ligation reaction

• Self-circularization of Linear DNA (with T4 DNA Ligase) (Fermentas)
    
• Linker Ligation (with T4 DNA Ligase) (Fermentas)
    
• DNA Insert Ligation into Vector DNA (with T4 DNA Ligase) (Fermentas)

Others

• Generating New Cleavage Sites (NEB)
  • Cleavage with two restriction endonucleases that produce compatible overhangs.
      

·         (NEB)
Presents quick reference table for application of modifying enzymes.

·         (NEB)

·         (LTI)
What are the best conditions for double digests? 
What buffer do you recommend for double digests?
How far from the end of a DNA fragment will a specific enzymes can digest? and more...  

·         (Donis Keller Lab)
Buffer constitution for usr/localious enzymes

• (Donis Keller Lab)
  To retrieve and purify any specific DNA fragment from an agarose gel slice; the expected yield is from 50-75% of the amount in the gel slice.
    
• (Donis Keller Lab)
  Recovery of DNA from agarose gel
    
• Thermal Inactivation (Fermentas)
  A simple, reversible way to a stop restriction reaction is by adding EDTA, which chelates Mg²⁺, thereby preventing catalysis. If further manipulations of the digested DNA are to be performed, the restriction endonuclease should be inactivated. Phenol/chloroform extraction and ethanol precipitation is an irreversible method for inactivation and removal of all restriction endonucleases...