Minnesota/14 July 2008

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|1. '''Plasmid DNA Purification Using the QIAprep:''' Use 2mL LB cultures of ligation products from 07-11-2008. ''Procedure:''
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|'''a.''' Resuspend bacterial cells in 250uL Buffer P1 and transfer to microcentrifuge tube
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|'''b.''' Add 250 uL Buffer P2 and invert 4-6 times
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|'''c.''' Add 350uL Buffer N3 and mix immediately by inverting 4-6 times
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|'''d.''' Centrifuge for 10 minutes @ 13,000 rpm in a table-top centrifuge. White pellet will form (made up of cell debris).
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|'''e.''' Apply supernatants from step D to the QIA prep spin columns. Centrifuge for 30-60 seconds --> discard the flow through.
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|'''f.''' Wash the QIA prep spin column  by adding 0.5mL (500uL) Buffer PB. Centrifuge for 30-60 seconds. Discard the flow through.
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|'''g.''' Wash QIA prep spin column by adding 0.75mL (750uL) Buffer PE. Centrifuge for 30-60 seconds. Discard the flow through.
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|'''h.''' Centrifuge for additional 1 minute to remove residual buffer.
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|'''i.''' Place the QIA prep spin column in a clean 1.5mL  centrifuge tube. To elute DNA, add 50uL of Buffer EB (10mM Tris-Cl, pH 8.5) to center of spin column and centrifuge.

Revision as of 16:21, 14 July 2008

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1. Plasmid DNA Purification Using the QIAprep: Use 2mL LB cultures of ligation products from 07-11-2008. Procedure:
a. Resuspend bacterial cells in 250uL Buffer P1 and transfer to microcentrifuge tube
b. Add 250 uL Buffer P2 and invert 4-6 times
c. Add 350uL Buffer N3 and mix immediately by inverting 4-6 times
d. Centrifuge for 10 minutes @ 13,000 rpm in a table-top centrifuge. White pellet will form (made up of cell debris).
e. Apply supernatants from step D to the QIA prep spin columns. Centrifuge for 30-60 seconds --> discard the flow through.
f. Wash the QIA prep spin column by adding 0.5mL (500uL) Buffer PB. Centrifuge for 30-60 seconds. Discard the flow through.
g. Wash QIA prep spin column by adding 0.75mL (750uL) Buffer PE. Centrifuge for 30-60 seconds. Discard the flow through.
h. Centrifuge for additional 1 minute to remove residual buffer.
i. Place the QIA prep spin column in a clean 1.5mL centrifuge tube. To elute DNA, add 50uL of Buffer EB (10mM Tris-Cl, pH 8.5) to center of spin column and centrifuge.
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