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 on
   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]

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

Back to Main