Team:Columbia-Cooper-NYC/Miniprep
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= Plasmid Isolation Protocol = | = Plasmid Isolation Protocol = | ||
+ | * Following is a plasmid isolation protocol provided by QIAGEN: | ||
+ | ° µl | ||
+ | # Resuspend pelleted bacterial cells in 250µl Buffer P1 and transfer to a microcentrifuge tube | ||
+ | # Add 250µl Buffer P2 and mix thoroughly by inverting the tube 4-6 times | ||
+ | # Add 350µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times | ||
+ | # Centrifuge for 10 min. at 13,000 rpm in a table-top microcentrifuge | ||
+ | # Apply the supernatant (from step 4) to the QIAprep spin column by decanting | ||
+ | # Centrifuge for 30-60 seconds and discard the flow-through | ||
+ | # Wash QIAspin column by adding 0.75ml Buffer PE and centrifuging for 30-60 seconds | ||
+ | # Discard the flow-through, and centrifuge for an additional 1-3 minutes to remove residual wash buffer | ||
+ | # To elute DNA, place the QIAprep column in a clean 1.5ml microcentrifuge tube. Add 50µl water to center of QIAprep spin column, let stand for 1 minute and centrifuge for 1-3 minutes. |
Revision as of 23:53, 1 October 2012
Plasmid Isolation Protocol
- Following is a plasmid isolation protocol provided by QIAGEN:
° µl
- Resuspend pelleted bacterial cells in 250µl Buffer P1 and transfer to a microcentrifuge tube
- Add 250µl Buffer P2 and mix thoroughly by inverting the tube 4-6 times
- Add 350µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times
- Centrifuge for 10 min. at 13,000 rpm in a table-top microcentrifuge
- Apply the supernatant (from step 4) to the QIAprep spin column by decanting
- Centrifuge for 30-60 seconds and discard the flow-through
- Wash QIAspin column by adding 0.75ml Buffer PE and centrifuging for 30-60 seconds
- Discard the flow-through, and centrifuge for an additional 1-3 minutes to remove residual wash buffer
- To elute DNA, place the QIAprep column in a clean 1.5ml microcentrifuge tube. Add 50µl water to center of QIAprep spin column, let stand for 1 minute and centrifuge for 1-3 minutes.