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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

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.