Daniel:Protocols/EtOHPrecip: Difference between revisions

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=Ethanol Precipitation=
=Ethanol Precipitation=
[[Daniel Jacobsen|Back to Main]]


   Purpose:  To purifiy DNA.
   Purpose:  To purifiy DNA.


==Protocol==
==Protocol==
   1. Add (X=volume sample):
   1. Add:
    At least 2.5X 100% Ethanol
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
    0.1X 3M NaOAc
|- style="background-color:#EEECE1;font-size:12pt;font-weight:bold" align="center"
    1/300X glycoblue (co-precipitate, helps you see the DNA)
| 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
   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 750 uL chilled 75% EtOH
   5. Spin at 10000 rpm for 10 minutes at 4 C
   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
|}
[[Daniel Jacobsen|Back to Main]]

Latest revision as of 18:33, 2 July 2014

Ethanol Precipitation[edit]

Back to Main

 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

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