Purification of DNA: Difference between revisions
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#[[Isopropanol Precipitation for PCR Purification]] | #[[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 | #*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! [[User:Jasu|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. | |||
==Filtration / affinity columns== | ==Filtration / affinity columns== |
Revision as of 04:15, 25 April 2007
back to protocols | ||
There are several methods for purifying DNA. The one you choose depends on the nature of your DNA sample and your downstream applications.
Alcohol 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.
Filtration / affinity columns
- Centrifugal filtration/Nucleic acids
- for small DNA fragments (~50-200 bp in length)
- removes proteins, nucleotides and salts
- Miniprep
- for purifying plasmid DNA from E. coli cells
- QIAquick PCR purification
- for typical DNA fragments (> 200 bp in length)