Team:Newcastle/PCR purification

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(Protocol)
(Materials)
 
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==Materials==
==Materials==
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* 1.5ml microcentrifuge tubes
+
* 1.5 ml microcentrifuge tubes
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* 2ml collection tube
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* 2 ml collection tube
* QIAquick columns
* QIAquick columns
* Qiagen Buffer PB
* Qiagen Buffer PB
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# Discard flow-through. Add 0.75 ml of Buffer PE to the spin column to wash and centrifuge for 30 to 60 seconds at 13,000 rpm.
# Discard flow-through. Add 0.75 ml of Buffer PE to the spin column to wash and centrifuge for 30 to 60 seconds at 13,000 rpm.
# Discard flow-through and centrifuge for another 1 minute at 13,000 rpm.
# Discard flow-through and centrifuge for another 1 minute at 13,000 rpm.
-
# Place QIAquick spin column into a clean 1.5ml microcentrifuge tube.
+
# Place QIAquick spin column into a clean 1.5 ml microcentrifuge tube.
# Add 50 µl Buffer EB to the center of the membrane of spin column and centrifuge for 1 minute at 13,000 rpm.
# Add 50 µl Buffer EB to the center of the membrane of spin column and centrifuge for 1 minute at 13,000 rpm.
# If analysing purified DNA using gel, add 1 volume of Loading Dye to 5 volumes of DNA.
# If analysing purified DNA using gel, add 1 volume of Loading Dye to 5 volumes of DNA.
 +
# Store the PCR product either at 4°C or at -20°C.

Latest revision as of 14:41, 26 October 2010

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PCR Purification

Materials

  • 1.5 ml microcentrifuge tubes
  • 2 ml collection tube
  • QIAquick columns
  • Qiagen Buffer PB
  • Qiagen Buffer EB
  • Qiagen Buffer PE
  • DNA mixture from PCR

Protocol

  1. Add 5 volumes of Qiagen Buffer PB to 1 volume of PCR product.
  2. Put a QIAquick spin column into a 2ml collection tube.
  3. Apply the mixture to the QIAquick column to bind the DNA and centrifuge for 30 to 60 seconds at 13,000 rpm.
  4. Discard flow-through. Add 0.75 ml of Buffer PE to the spin column to wash and centrifuge for 30 to 60 seconds at 13,000 rpm.
  5. Discard flow-through and centrifuge for another 1 minute at 13,000 rpm.
  6. Place QIAquick spin column into a clean 1.5 ml microcentrifuge tube.
  7. Add 50 µl Buffer EB to the center of the membrane of spin column and centrifuge for 1 minute at 13,000 rpm.
  8. If analysing purified DNA using gel, add 1 volume of Loading Dye to 5 volumes of DNA.
  9. Store the PCR product either at 4°C or at -20°C.


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