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From 2012.igem.org

Contents 1 I. Digest pBAV1K-T5-gfp Plasmid and Cassette with XbaI and PstI 2 II. QIAquick PCR Purification Protocol 3 III. Digest Restriction-digested Plasmid with CIP 4 IV. QIAquick PCR Purification Protocol 5 V. Measure the Concentration of Extracted DNA 5.1 RESULTS

I. Digest pBAV1K-T5-gfp Plasmid and Cassette with XbaI and PstI

• Mix (plasmid):

• 37.5 uL water
• 5 uL pBAV1K-T5-gfp DNA (~1 ug at 200 ng/uL)
• 0.5 uL 100x BSA
• 5 uL 10 NEB Buffer 3
• 1 uL XbaI
• 1 uL PstI

• Mix (cassette):

• 10.32 uL water
• 32.18 uL cassette DNA (~1 ug at 31.075 ng/uL)
• 0.5 uL 100x BSA
• 5 uL 10 NEB Buffer 3
• 1 uL XbaI
• 1 uL PstI

• Incubate tubes in 37 ^oC water bath for 4 hours

II. QIAquick PCR Purification Protocol

• Add 5 volumes Buffer PB to 1 volume of reaction, mix

Reaction	Volume PB
plasmid - 50 uL	250 uL
cassette - 50 uL	250 uL

• Note: The solutions were all yellow, OK to proceed

• Apply samples to QIAquick columns, centrifuge for 1 minute at 13000 rpm, discard flow-through

• Add 750 uL of Buffer PE to the QIAquick column, centrifuge for 1 min and discard flow through

• Centrifuge again for 1 minute

• Transfer the column to clean microcentrifuge tube

• Elute cassette DNA by adding 30 uL of Buffer EB to the center of the column and let stand for 4 minutes

• Elute plasmid DNA by adding 30 uL of water to the center of the column and let stand for 4 minutes

• Centrifuge for 1 minute, store cassette DNA at 4 ^oC

III. Digest Restriction-digested Plasmid with CIP

• Mix:

• 28 uL of plasmid DNA (assume ~1 ug)
• 16 uL water
• 5 uL 10X NEB Buffer 3
• 1 uL diluted CIP

• 1 uL of CIP stock into 10 uL of water

• Incubate in 37 ^oC water bath for 1 hour

IV. QIAquick PCR Purification Protocol

• Add 5 volumes Buffer PB to 1 volume of reaction, mix

Reaction	Volume PB
plasmid - 50 uL	250 uL

• Note: The solutions were all yellow, OK to proceed

• Apply sample to QIAquick column, centrifuge for 1 minute at 13000 rpm, discard flow-through

• Add 750 uL of Buffer PE to the QIAquick column, centrifuge for 1 min and discard flow through

• Centrifuge again for 1 minute

• Transfer the column to clean microcentrifuge tube

• Elute olasmid DNA by adding 30 uL of Buffer EB to the center of the column and let stand for 4 minutes

• Centrifuge for 1 minute, store plasmid DNA at 4 ^oC

V. Measure the Concentration of Extracted DNA

• Bring samples and Buffer EB bottle upstairs to Take3 plate reader
• Add 2 uL of each sample according to the following chart:

Blank (EB)	Blank
Blank	Blank
plasmid	cassette

• Use the program to blank the machine and measure sample concentrations

RESULTS

Concentration:

Sample	Concentration (ng/uL)	Approx. volume (uL)
plasmid	19.714	~28
cassette	15.692	~28