Lab Protocols: Difference between revisions
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===Phenol/chloroform extraction=== | ===Phenol/chloroform extraction=== | ||
Complete removal of protein, doesn't change buffer. Typically followed by ethanol precipitation. Has to be handle carefully in order not to get phenol contamination, which will inhibit any enzymatic reaction. | Complete removal of protein, doesn't change buffer. Typically followed by ethanol precipitation. Has to be handle carefully in order not to get phenol contamination, which will inhibit any enzymatic reaction. | ||
Protocol: | |||
To the sample you want to purify, add 0.1 volume of 3M NaOAc (pH=5.2), 2.5 volume of 100% ethanol. | |||
[OPTIONAL] To visualize the DNA palettes, Glycogen or Linear Acrylamide or GlycoBlue can be added. | |||
Freeze the tube at -20C or -75C for > 20min. | |||
Spin the tube at >10000xg for 15 min 4C | |||
Carefully remove all liquid, add 750ul 75% Ethanol, vortex for > 30 sec (or mix by pipetting) | |||
Spin the tube at >7500xg for 5min at 4C | |||
Carefully remove all liquid, air dry for 5min | |||
Resuspend DNA in appropriate buffer or water. | |||
===Ethanol Precipitation=== | ===Ethanol Precipitation=== | ||
The most robust purification method. Works for samples of any volume. Good for single-standed and double-stranded DNA across a wide range of size. Can not be used to remove short oligos. Purification takes longer (~1 hour) than column-based method. Difficult to quantify the precipitated DNA with spectrophotometer if co-precipitants (Glycogen, linear acrylamide) are used. | The most robust purification method. Works for samples of any volume. Good for single-standed and double-stranded DNA across a wide range of size. Can not be used to remove short oligos. Purification takes longer (~1 hour) than column-based method. Difficult to quantify the precipitated DNA with spectrophotometer if co-precipitants (Glycogen, linear acrylamide) are used. |
Revision as of 18:22, 25 February 2008
Purification & buffer exchange
Phenol/chloroform extraction
Complete removal of protein, doesn't change buffer. Typically followed by ethanol precipitation. Has to be handle carefully in order not to get phenol contamination, which will inhibit any enzymatic reaction.
Protocol: To the sample you want to purify, add 0.1 volume of 3M NaOAc (pH=5.2), 2.5 volume of 100% ethanol. [OPTIONAL] To visualize the DNA palettes, Glycogen or Linear Acrylamide or GlycoBlue can be added. Freeze the tube at -20C or -75C for > 20min. Spin the tube at >10000xg for 15 min 4C Carefully remove all liquid, add 750ul 75% Ethanol, vortex for > 30 sec (or mix by pipetting) Spin the tube at >7500xg for 5min at 4C Carefully remove all liquid, air dry for 5min Resuspend DNA in appropriate buffer or water.
Ethanol Precipitation
The most robust purification method. Works for samples of any volume. Good for single-standed and double-stranded DNA across a wide range of size. Can not be used to remove short oligos. Purification takes longer (~1 hour) than column-based method. Difficult to quantify the precipitated DNA with spectrophotometer if co-precipitants (Glycogen, linear acrylamide) are used.
Column purification
Qiagen's Qiaquick column
Complete removal of protein and buffer. Good for both single stranded and double stranded DNA. Elution volume is slightly larger than MinElute column. Good for general purpose purification. More expensive than Bioneer's AccuPrep column.
Qiagen's MinElute column
Complete removal of protein and buffer. Small elution volume (10ul), great to obtain highly concentrated DNA. Yield is slightly lower than Qiaquick.
Bioneer's AccuPrep column
Complete removal of protein and buffer. Low cost, good for double stranded DNA. Questionable for single-stranded DNA.
Size exclusion purification
- Sephadex G25, G50
Size selection column
- Millipore: YM-10, YM-30, YM-50, YM-100