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Team:Newcastle/PCR purification
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(New page: =PCR Purification= ==Materials== * 1.5ml microcentrifuge tubes * 2ml collection tube * QIAquick columns * Buffer PB * Buffer EB * Buffer PE * DNA mixture from PCR ==Protocol== # Add 5 ...) |
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=PCR Purification= | =PCR Purification= | ||
==Materials== | ==Materials== | ||
| - | * 1. | + | * 1.5 ml microcentrifuge tubes |
| - | * | + | * 2 ml collection tube |
* QIAquick columns | * QIAquick columns | ||
| - | * Buffer PB | + | * Qiagen Buffer PB |
| - | * Buffer EB | + | * Qiagen Buffer EB |
| - | * Buffer PE | + | * Qiagen Buffer PE |
* DNA mixture from PCR | * DNA mixture from PCR | ||
==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. | + | # 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. | + | # 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. | ||
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| + | '''Go back to our [[Team:Newcastle/Protocol list|Protocol List]]''' | ||
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| + | {{Team:Newcastle/footer}} | ||
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
- 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 1.5 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.
- 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.
Go back to our Protocol List
   
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