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From 2011.igem.org

Contents 1 Restriction Enzyme Double Digest 1.1 Materials 1.2 Buffer Compatibility Chart 1.3 Procedure 2 Gel Extraction/Purification Procedure 2.1 Materials 2.2 Procedure 3 Transformations 3.1 Materials 3.2 Procedure 4 Liquid Cultures 4.1 Materials 4.2 Procedure 5 PCR 5.1 Mix

Restriction Enzyme Double Digest

Materials

• 22 uL dH2O
• 1 uL BSA
• 5 uL Buffer
• 20 uL Template
• 1 uL Enzyme 1
• 1 uL Enzyme 2

Buffer Compatibility Chart

	1	2	3	4
EcoRI	100	100	100	100
SpeI	75	100	25	100
PstI	75	75	100	50
NheI	100	100	10	100
XbaI	0	100	75	100

Procedure

• Mix reactants thoroughly.
• Place at 37 C for 3 hours.
• Increase to 80 C for 20 minutes to kill enzymes (some enzymes need only a 65 C heatkill, check enzyme).
• Run on a gel and extract product.

Gel Extraction/Purification Procedure

Materials

• GeneJET Gel Extraction Kit
• Binding Buffer (1 uL for every mg of agarose gel)
• 700 uL of Wash Buffer
• 50 uL of Elution Buffer

Procedure

• Add the binding buffer to the gel slice in a microcentrifuge tube.
• Incubate the gel mixture at 50-60 °C for 10 minutes (until melted).
• Transfer the solution to a GeneJET purification column.
• Centrifuge for 30-60 seconds at 12000 x g and discard the flow through.
• Add Wash Buffer and centrifuge for 1 minute.
• Discard flow through, then centrifuge empty column for 1 minute.
• Transfer the column into a fresh 1.5 ml microfuge tube.
• Add Elution Buffer.
• Centrifuge for 1 minute and collect the flow-through.

Transformations

Materials

• Competent cells
• DNA template
• 800 uL of LB
• LB+antibiotic plates

Procedure

• Thaw competent cells on ice.
• Transfer 50 uL of competent cells to chilled falcon tubes.
• Add 1 uL of template to cells (2.5 uL if dilute).
• Incubate on ice for 30 minutes.
• Heat schock in 42 °C water bath for 90 seconds.
• Immediately place back onto ice for 2 minutes.
• Add 800 uL of LB to each tube.
• Incubate at 37 °C for 1 hour.
• Place 200 uL of the transformed cells on plates containing LB and the appropriate antibiotic.
• Incubate overnight at 37 °C.

Liquid Cultures

Materials

• LB
• Plated colonies of cells
• Antibiotic stock

Procedure

• In a sterile environment, add 4 mL of LB to each falcon tube.
• Add the appropriate amount of antibiotic.
  • for carb, add 8 uL.
• With a tip, scoop a colony and place it in the falcon tube.
• Incubate overnight at 37 °C.

PCR

Mix

• 10ul Q solution
• 5ul 10x buffer
• 1.25ul DNTPs
• 1ul Forward primer
• 1ul Reverse primer
• 1ul Template
• .3ul Taq
• .15ul PFU
• 30ul dH2O