digestion and labelling of large ammount of DNA - (Jul/21/2012 )

Hello ,I want to apply this protocol to produce fluorescent labelled restriction fragment:

1. Digest 50µg of PhiX174 DNA with DdeI and CfoI endonuclease in 200µl reaction).

2. Following the restriction digest, add
80 µl dATP, dCTP, dGTP, (2.5 mM each)
8 µl of 1 mM 12-Texas Red dUTP (Molecular Probes), or any other dUTP or dTTP with a fluorescent label
8 µl Klenow Fragment of DNA Polymerase (40 units)


my question is: can be the concentration of reagent be optimezed?
For example is necessary to perform the digestion in 200ul of reaction?How many ul reaction are needed to digest 50ug of phage dna?

Thank you for any help
Vincenzo

Hi,

If your question is current. You can calculate the minimal volume of RE reaction as follows: Theoretically one unit of DdeI is defined (according to New England Biolabs) as the amount of enzyme required to digest 1 µg of λ DNA in 1 hour at 37°C in a total reaction volume of 50 µl. Practically you sould use much more enzyme, I would first try to use 50 U for overnight for 50 µg PhiX174 DNA. 50 U DdeI is in 5 µl storage volume (in DdeI produced by New England Biolabs). The volume of enzyme must be maximally 10% of the total volume because of star activity, therefore, the minimal volume of the reaction should be 50 µl in this case. Otherwise, you won't make a mistake, if you use a larger total volume. If you want to use more concentrated DNA for the labelling step, you can extract the DNA with a kit, and then, DNA can be solved again in a smaller volume.

Marton Doleschall, PhD
human molecular genetics
http://www.kutlab.hu/doleschall.php

thank you very much!