Digestions

Overview
Digestions
pGT4ΔB
Electrophoresis
DNA clean-up
Ligation
Fragment isolation
Competent cells
Transformation
Recombinants
pGTλ3758ΔH
Miniprepping
Blotting
Probe Labeling
Hybridisation
Probe Detection
PCR

Setting-up digestions
Using a Mastermix
What can go wrong?

 

 

Restriction enzyme digestions

Restriction enzyme digestions are performed by incubating double-stranded DNA molecules with an appropriate amount of restriction enzyme, in its respective buffer as recommended (and often supplied) by the commercial supplier, and at the optimal temperature for that specific enzyme.
One enzyme unit  is defined as the amount of enzyme needed to completely digest one microgram of linear double-stranded DNA in one hour at the appropriate temperature.To cut covalently closed circular (ccc) plasmid DNA and for unpure DNA preparations more enzyme is needed. Usually, an (up to 10 times) excess of enzyme is used.
In general for DNA analysis, you digest 0.2 to 2μg per digest (a minimum amount of approximately 20 ng per DNA band is needed for visualisation in an EthBr-containing gel).
The digestion reactions usually are incubated for 1-3 hours, to ensure complete digestion, at the optimal temperature for enzyme activity, typically 37 C.

 

  See the Movie about
some commonly used restriction enzymes

Read the Movie Text

 

 

Setting-up digestions

1 Check the concentration of your DNA stocks (they are usually in your box in the -20 C freezer) and decide how much you want to digest.  

In general: maximally 2 μg per 20 μl reaction mixture.
For an analytical gel: 20 to 200 ng per expected DNA band.
For a preparative gel (i.e. to purify DNA fragments  from bands): up to 1μg per expected band.

2 Check the concentrations of the restriction enzymes to be used. They are usually stored in the central enzymes -20 C freezer in the lab; concentration is mostly between 2 and 10 units/μl.  

Glycerol concentrations higher than 5% inhibit restriction enzymes.
So, do not use more than 2μl enzyme stock solution(s) in the 20 μl of reaction mixture, since enzyme stocks contain 50% glycerol.

3 Check the concentration of the available Digestion Buffer stock solution; usually 5x or 10x concentrated.  
4 Draw a pipetting scheme for your digestion reactions. Typically: you will set-up 20μl reactions with 2 to 10 units/μg DNA.  
5 Mark your tubes, typically 1.5ml disposable tubes, fill them, mix well, collect by a few seconds centrifugation and start the reaction by placing them in a 37 C waterbath.  

 

Some helpfull tips:

  • Keep restriction enzyme stock solution always in ice; their activity will gradually decrease after successive exposures to higher temperature.

  • Add water first, enzyme(s) last when making up multiple digestions with the same enzyme(s), it's good to make a stock mix ("mastermix"), see below.

  • When adding very small volumes, e.g. less than 5μl, pipet a few times up-and-down to transfer all of the liquid from the tip. This is especially important when adding the very viscous (50% glycerol!) enzyme stock solutions.

  • When you've touched any liquid already in the tubes, change the pipet tip. 

 

Setting-up multiple digestions using the same enzymes: make a Mastermix!

A very convenient and accurate way of starting a series of reactions (e.g. digestions, PCR) is to make a so-called mastermix: a relatively large amount of mixture that contains everything-but-one-component of the digestion. If you for example would have to digest a large number of minipreps with the same enzyme(s), the best way to do this is as follows:
make a large volume of a mixture containing water, concentrated digestion buffer and enzymes, fill the tubes with an appropriate amount of this mastermix and add a small amount of the minipreps to the tubes to start the digestion.

 

What can go wrong?

When not all DNA molecules are fully cut by the restriction enzyme(s), some cutting sites in some of the molecules stay intact. This may result in more DNA fragments than expected. Such incomplete (or: "partial") digestion can occur for many reasons:

  • bad quality or an insufficient amount of restriction enzyme
  • inaccurate pipetting when handling very small volumes
  • bad quality of the DNA preparation, i.e. unpure DNA; impurities may prevent optimal restriction enzyme activity
  • wrong buffer and temperature conditions during the reaction
     

When linearisation of a plasmid by a single cut was only partial, the agarose gel will show some OC and CCC form plasmid after such an incomplete digestion (see About plasmid DNA and gel electrophoresis in the Electrophoresis section)...

Sometimes, deliberately partial digestions are used to generate a particular DNA fragment.
 

See the Movie about Partial Digestions: Part A and Part B
Read the Movie Text