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#1 amarieu

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Posted 19 December 2010 - 09:00 PM

Hi All-

How do you decide which Restriction Enzymes to do a Digestion with?
I am new to the field and I am trying to take on a project but I am unsure how to decide which enzymes to digest the mini prep DNA with to check that it worked.

Any help at all would be GREAT!

Thanks.

#2 protolder

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Posted 19 December 2010 - 10:50 PM

Hola, When you want clone you have to choose RE of the MCS of the vector that donīt cut the insert, and ligate this with the insert from PCR or other vectos with the same ends, making a directional cloning. Analyzing clones you cut with the same enzymes to see the insert rescued and other enzymes that cut vector and insert to give the teorical fragments that you have calculate in an scheme map of your recombinant clone. Buena suerte

#3 perneseblue

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Posted 27 December 2010 - 07:26 PM

Well the criteria for selecting restriction enzymes for a diagnostic digest is
1- a DNA fragment pattern unique to the plasmid of interest
That mean, the restriction digest cut the plasmids into a few fragments. And the size of these fragments are unique to the plasmid you are working on. If you are building a plasmid, this mean that this fragment pattern is different from either the original empty vector or the plasmid which the DNA insert originally came from.
For example,
Your desired plasmid, when cut with BamHI and EcoRI will produce 3 bands.
The empty vector cut with BamHI/EcoRI will produce 1 band
The host of the insert will produce 2 bands.


2 - a fragment pattern that is easy to see and discriminate on a gel.
You must be able to tell the individual bands apart.
If the fragments are too close in size, all you will see is a single fat band.
As example, a DNA fragment pattern of 6000bp,. 5500bp and 2000bp will look like 2 bands rather than 3.
May your PCR products be long, your protocols short and your boss on holiday




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