Download Restriction Digest and Analysis of Plasmid

Restriction Digest and Analysis of Plasmid
Equipment:
Ice bucket
Microcentrifuge
Micropipetters
Electrophoreses Set
Starting Material:
miniature plasmid preparation
Reagents and disposable Materials:
1.5 ml microcentrifuge tubes
Micropipette tips
Restriction enzymes
Restriction enzymes buffers
sterile deionized water
Dnase-free Rnase
1 % agarose TAE gel
TAE running buffer
Restriction Digest and Analysis Procedure:
1) Keep and or thaw 40 ul miniature preparation of the plasmid on ice. Thaw restriction
enzyme buffers on ice but maintain restriction enzymes at -20 C until just prior to use.
2) For single enzyme digestions combine the following in a 1.5 ml microcentrifuge tube:
5 ul of miniature plasmid preparation DNA
4 ul of 10X restriction enzyme buffer (consult manufacturers information)
29 ul of sterile deionized water
1 ul of Dnase-free Rnase (to remove any residual RNA)
1 ul of restriction enzyme
or for double enzyme digestions combine the following in a 1.5 ml microcentrifuge tube
5 ul of miniature plasmid preparation DNA
4 ul of 10X restriction enzyme buffer (consult double digest information)
28 ul of sterile deionized water
1 ul of Dnase-free Rnase (to remove any residual RNA)
1 ul of the first restriction enzyme
1 ul of the second restriction enzyme
3) Mix and briefly spin to place sample at tube bottom.
4) Incubate @ 37 C for 1 hour (unless otherwise specified).
Can be stored @-20 C after this step.
5) Electrophorese in a 1 % agarose TAE gel. A range of 0.7 to 2 % agarose gels are often
used
6) Visualize and construct restriction map.
7) Select and excise band(s) for subsequent cloning.