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Team:Newcastle/PCR purification
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==Protocol== | ==Protocol== | ||
| - | # Add 5 volumes of Buffer PB to 1 volume of PCR product. | + | # Add 5 volumes of Qiagen Buffer PB to 1 volume of PCR product. |
# Put a QIAquick spin column into a 2ml collection tube. | # Put a QIAquick spin column into a 2ml collection tube. | ||
| - | # Apply the mixture to the QIAquick column to bind the DNA and centrifuge for 30 to 60 seconds. | + | # Apply the mixture to the QIAquick column to bind the DNA 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. | + | # 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. | + | # 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 1.5ml microcentrifuge tube. | ||
| - | # Add 50 µl Buffer EB to the center of the membrane of spin column and centrifuge for 1 minute. | + | # 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. | ||
Revision as of 12:28, 26 October 2010
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PCR Purification
Materials
- 1.5ml microcentrifuge tubes
- 2ml collection tube
- QIAquick columns
- Qiagen Buffer PB
- Qiagen Buffer EB
- Qiagen Buffer PE
- DNA mixture from PCR
Protocol
- Add 5 volumes of Qiagen Buffer PB to 1 volume of PCR product.
- Put a QIAquick spin column into a 2ml collection tube.
- Apply the mixture to the QIAquick column to bind the DNA 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.
- Place QIAquick spin column into a clean 1.5ml 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.
- If analysing purified DNA using gel, add 1 volume of Loading Dye to 5 volumes of DNA.
Go back to our Protocol List
   
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