Daniel:Protocols/EtOHPrecip: Difference between revisions
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==Protocol== | ==Protocol== | ||
1. Add | 1. Add: | ||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | ||
|- style="background-color:#EEECE1;font-size:12pt;font-weight:bold" align="center" | |- style="background-color:#EEECE1;font-size:12pt;font-weight:bold" align="center" | ||
Line 23: | Line 23: | ||
|} | |} | ||
where X = volume of the sample | |||
2. Incubate for at least 1 hour at -80 C | 2. Incubate for at least 1 hour at -80 C | ||
For smaller fragments it is helpful to incubate overnight | For smaller fragments it is helpful to incubate overnight | ||
3. Centrifuge at 4 C | 3. Centrifuge at 4 C | ||
4. Remove supernatant and add 500 uL chilled 75% EtOH | 4. Remove supernatant and add 500 uL chilled 75% EtOH | ||
5. | 5. Centrifuge at minutes at 4 C | ||
6. Dry the pellet in the hood | 6. Dry the pellet in the hood | ||
7. Resuspend pellet in desired volume ddH20 | 7. Resuspend pellet in desired volume ddH20 | ||
8. Store DNA at -20C | 8. Store DNA at -20C | ||
==Recommended Centrifuge Times== | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#EEECE1;font-size:12pt;font-weight:bold" align="center" | |||
| width="65" height="15" | Spin | |||
| width="65" | Size Tube | |||
| width="65" | Time | |||
| width="65" | RPM | |||
|- style="background-color:#8DB4E2;font-size:12pt" align="center" | |||
| align="center" height="15" | 1 | |||
| 15 mL | |||
| align="center" | 30 | |||
| align="center" | 3000 | |||
|- style="background-color:#BFBFBF;font-size:12pt" align="center" | |||
| align="center" height="15" | 1 | |||
| 1.5 mL | |||
| align="center" | 25 | |||
| align="center" | 12000 | |||
|- style="background-color:#8DB4E2;font-size:12pt" align="center" | |||
| align="center" height="15" | 2 | |||
| 1.5 mL | |||
| align="center" | 10 | |||
| align="center" | 10000 | |||
|} | |||
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Revision as of 23:32, 17 April 2013
Ethanol Precipitation
Purpose: To purifiy DNA.
Protocol
1. Add:
Chemical | Volume |
100% EtOH | ≥ 2.5X |
3 M NaOAc | 0.1X |
Glycoblue | 1/300X |
where X = volume of the sample
2. Incubate for at least 1 hour at -80 C For smaller fragments it is helpful to incubate overnight 3. Centrifuge at 4 C 4. Remove supernatant and add 500 uL chilled 75% EtOH 5. Centrifuge at minutes at 4 C 6. Dry the pellet in the hood 7. Resuspend pellet in desired volume ddH20 8. Store DNA at -20C
Recommended Centrifuge Times
Spin | Size Tube | Time | RPM |
1 | 15 mL | 30 | 3000 |
1 | 1.5 mL | 25 | 12000 |
2 | 1.5 mL | 10 | 10000 |