9/7/06—Module I –Ligation of Kanr Insert into Vector pSP64
PURPOSE: To perform and verify (by gel electrophoresis) the ligation of the kanr gene into vector pSP64.
MATERIALS:
Qualified water
Vector pSP64/purified kanr insert
T4 DNA Ligase reaction tubes
0.9% agarose gel or powdered agarose to make gel
1X Electrophoresis buffer (TBE)
Electrophoresis equipment
10X Loading dye
Molecular weight markers
Standard equipment—pipettes, tips, centrifuge vortex, etc
PROCEDURE:
1. Prep:
Set water bath to 16ºC.
Make 1X electrophoresis buffer (400 ml) from 5X TBE.
Make 0.9% agarose gel in min-gel apparatus.
Ice bucket w/ice
2. Label three1.5 ml µfuge tubes:
1—stock ligation mixture
2—ligation “+” control
3—ligation experimental reaction (kanr + pSP64)
3. Combine the ligation components to tube “1” in the following order:
Tube “A” à40 µl qualified water
Tube “B” à20 µl Vector and kanr frags
Mix by tapping or vortexing. Put on ice.
4. Transfer 20 µl from tube “1” to tube “2”. Cap tube “1”.
5. Add 5 µl of 10X gel loading dye to tube “2”, mix , and reserve.
6. Vortex the T4 DNA Ligase Reaction tube (tube “C”), and tap or spin to collect T4 ligase pellet at bottom of tube.
7. Transfer remaining stock ligation reaction from tube 1 to T4 DNA Ligase Reaction Tube. Re-label this tube “1”, incubate 5 m @RT.
8. Carefully mix the reaction by gently pipetting up and down. Spin briefly to collect liquid.
9. Incubate @ 16ºC for 30m. Mix by tapping periodically.
10. Transfer 20 µl from tube “1” to tube “3”. Add 5 µl 10X gel loading dye to tube “3”. Store tube “1” in the -20ºC freezer.
11. Electrophorese the contents of tubes “2” and “3”. Include 1 kb Ladder as marker (5µl in 20 µl 1 X gel loading dye)
Voltage_____________
Time_______________
12. Stain in EtBr
[EtBr]_______________
Stain time____________
Destain time___________
RESULTS: see gel.