Purification of DNA: Difference between revisions

#[[QIAquick PCR purification]]

#*for typical DNA fragments (> 200 bp in length)

Products from the restriction need to a number of reagents removed from the previous restriction.  These include salts (from buffers) and restriction enzymes.  DNA can be removed and washed from solution by using column purification kits.  Here is a rough description of how the kit works.

Step 5: Elution.  Elution buffer is Tris buffer with some EDTA at a pH of 8-8.5.  EDTA binds to divalent cations, particularly magnesium (Mg2+). Tris has labile protons with a pKa of 8.30 (at 20 °C; this declines approximately 0.03 units per degree Celsius rise in temperature). Tris is often used when working with nucleic acids. Tris is an effective buffer for slightly basic solutions, which keeps DNA deprotonated and soluble in water. These ions are necessary co-factors for many enzymes; Magnesium is a co-factor for many DNA-modifying enzymes. Tris is toxic to mammalian cells, and reacts strongly with pH electrodes. It is a primary amine, and can thus react with aldehydes.