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From 2012.igem.org

Contents 1 I. Ligate Digested Plasmid and Digested Cassette Together 2 II. Transformation of DH5α Cells with pBAV1K-cassette 3 Lab Notes, Sunday, 12 Aug 2012 3.1 I. Check Plates for Growth 3.2 II. Streak Out WT ADP1

I. Ligate Digested Plasmid and Digested Cassette Together

• 1:1 vector:insert ratio - Mix:

• 0.34 uL water
• 2 uL 10X T4 Buffer
• 6 uL plasmid DNA (~0.05 pmol)
• 10.66 uL cassette DNA (~0.05 pmol)
• 1 uL T4 Ligase

• 1:3 vector:insert ratio - Mix:

• 1.2 uL water
• 2 uL 10X T4 Buffer
• 2.5 uL plasmid DNA (~0.0208 pmol)
• 13.30 uL cassette DNA (~0.0624 pmol)
• 1 uL T4 Ligase

• Incubate at room temperature for 10 minutes, then keep on ice

II. Transformation of DH5α Cells with pBAV1K-cassette

• Thaw 5 x50 uL competent DH5α cells on ice
• Add 4 uL of DNA/water according to the following table, swirl gently with pipette

Name	DNA
(-)	water
1:1t	1:1 ligation
1:1L	1:1 ligation
1:3t	1:3 ligation
1:3L	1:3 ligation

• Incubate tubes on ice for 30 minutes
• Heat pulse tubes in 42 ^oC water bath for 40 seconds
• Incubate on ice for 2 minutes
• Transfer cells to 11 mL culture tubes containing 500 uL of LB each, mix gently
• Incubate for an hour at 34 ^oC with shaking
• Spread with glass beads 100 uL of each culture on an LB agar plate according to the following table and incubate overnight at 34 ^oC

Name	Plate
(-)	LB + Tetracycline
1:1t	LB + Tetracycline
1:1L	LB
1:3t	LB + Tetracycline
1:3L	LB

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Lab Notes, Sunday, 12 Aug 2012

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I. Check Plates for Growth

RESULTS:

Growth on positive controls was a large number of colonies but not a lawn as expected. No growth on any of the Tet50 plates.

II. Streak Out WT ADP1

• Streak ADP1 from glycerol stock onto an LB plate
• Incubate at 34 ^oC overnight