Streptomyces:Protocols/DNA Digest: Difference between revisions

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Latest revision as of 18:14, 9 October 2007

Protocols - DNA Digest

Streptomyces @ UEA

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DNA Digest

Description
The fragmentation of DNA using restriction endonucleases (REs).

Approx. Duration:
Preparation ~5 minutes
Setting up the digest ~10 minutes
Whole Protocol ~1 hour 15 minutes


Uses
Analysis of plasmid, cosmid or genomic DNA in combination with various methods, e.g. Southern Blots, cloning, checking mini preps. Visualisation is achieved by running the/a sample of the digest on a agarose gel.


Prerequisites
Ideally you should have access to the sequence of the DNA you wish to analyse, if not a map should suffice. This will help make sense of the restriction pattern when run on an agarose gel and/or isolating a specific fragment for sub-cloning.


Safety
General laboratory & molecular biology safety rules apply.


Requirements
dH2O, DNA, Restriction Endonuclease (10U/µL) and its appropriate (10x) Buffer, incubator (i.e. a water bath) @ 37°C, ice, micro-centrifuge tube(s) (MCT) Optionally: Ribonuclease A (RNase), Alkaline Phosphatase (ALP), incubator @ 65°C.


Preparation
Thaw all the samples on ice, except the enzymes which are removed when needed. Label all tubes and ensure the incubator is ready.


Method
Add the ingredients in order, to a MCT and place in the incubator @ 37°C for 1 hour (3 hours - overnight for larger digests)

Total Volume - 20µL:
dH2O 16µL
Buffer 2µL
DNA 1µL
RE 1µL
Optional:
RNase 1µL
ALP 1µL


Example of a larger digest (volume of DNA depends on its concentration)

Total Volume - 200µL:
dH2O 173µL
Buffer 20µL
DNA 5µL
RE 2µL
Optional:
RNase 1µL
ALP 1µL


Notes
Remove the enzyme(s) from the freezer when needed and keep all the other ingredients on ice. Restriction enzymes are measured in units; 1 unit will completely digest 1µg of λDNA in 1 hour, under optimal conditions. Double digests are possible but you will need to consult the manufacturers documentation on which buffer is most compatible for both enzymes. The addition of RNase might be considered if a small DNA fragment is expected that the RNA may otherwise obscure. Alkaline Phosphatase is used when a fragment is cut from a plasmid/cosmid to prevent the DNA from re-ligating; the phosphate group on the 5' end is removed to prevent this. Another method to prevent re-ligation is to heat the MCT post-digest to 65°C for 3 minutes then transfer to ice.



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