Difference between revisions of "Purification of DNA"
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Latest revision as of 18:55, 29 December 2011
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There are several methods for purifying DNA. The one you choose depends on the nature of your DNA sample and your downstream applications.
- DNA Precipitation
- Ethanol precipitation of nucleic acids
- Ethanol precipitation of small DNA fragments
- Isopropanol Precipitation for PCR Purification
- use to concentrate DNA from PCR when you have verified that only a single PCR product exists. When performed properly this procedure exceeds the recovery of QIAquick PCR purification columns
2-propanol (isopropanol) precipitation yields less DNA than EtOH precipitation. (In my hands 60% less on average! Jasu) 2-propanol at RT reduces the risk of co-precipitation of salts (which can interfere with downstream experiments). 2-propanol is preferred for large volumes of DNA since less alcohol is required, plus it's faster since it doesn't require a cooling step. Glassy pellets from 2-propanol precipitation are harder to see than fluffy, salt-containing pellets from ethanol precipitation. 2-propanol pellets are also more loosely attached after centrifugation. Take care when decanting.
Size-dependent precipitation by PEG8000/MgCl2
A very easy and fast way to remove e.g. primers for downstream applications
Filtration / affinity columns
- Centrifugal filtration/Nucleic acids
- for small DNA fragments (~50-200 bp in length)
- removes proteins, nucleotides and salts
- for purifying plasmid DNA from E. coli cells
- 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 1: Enhance DNA negative charge. DNA is negatively charged because of the phosphate backbone, so this step enhances the negative charge. In the Qiagen kit, the PB solution contains guanine chloride, a protein detergent and denaturant. The solution is also slightly acidic due to the low pH
Step 2: Stick the negative DNA to the positively charged resin. Load your negatively charged DNA product into the tube and centrifuge it (usually around 14,000 rpm for 1 min).
Step 3: Wash DNA to remove small pieces of DNA. This is achieved using Ethanol and Tris buffer and is labelled as the “PE” buffer in the Qiagen kit. This solution solubilizes smaller pieces of DNA so that they can be eluted from the column. Generally you add a large amount of this stuff and spin the column (~750mL of PE buffer, spin for 1 min).
Step 4: Remove excess EtOH. After dumping out the EtOH from the previous step, another spin is good to remove all the EtOH from the column and DNA so that it doesn't get into the final eluted product. Spin for about 12 minutes at 14,000 rpm).
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.
DNA is now stabilized and ready for long-term storage at -20C.