Daniel:Protocols/EtOHPrecip: Difference between revisions
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=Ethanol Precipitation= | =Ethanol Precipitation= | ||
[[Daniel Jacobsen|Back to Main]] | |||
Purpose: To purifiy DNA. | |||
==Protocol== | |||
1. Add: | |||
{| 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="16" | Chemical | |||
| width="65" | Volume | |||
|- style="background-color:#EEECE1;font-size:12pt" align="center" | |||
| height="16" | 100% EtOH | |||
| ≥ 2.5X | |||
|- style="background-color:#EEECE1;font-size:12pt" align="center" | |||
| height="16" | 3 M NaOAc | |||
| 0.1X | |||
|- style="background-color:#EEECE1;font-size:12pt" align="center" | |||
| height="16" | 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 750 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== | |||
{| 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 | |||
|} | |||
[[Daniel Jacobsen|Back to Main]] |
Latest revision as of 18:33, 2 July 2014
Ethanol Precipitation[edit]
Purpose: To purifiy DNA.
Protocol[edit]
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 750 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[edit]
Spin | Size Tube | Time | RPM |
1 | 15 mL | 30 | 3000 |
1 | 1.5 mL | 25 | 12000 |
2 | 1.5 mL | 10 | 10000 |