Transformation of I15010 with DH5-α
The I15010 is transformed with competent cell DH5-α.
- Fill up the box with ice.
- Take 1 eppendorf tube,DH5-α from the -80°C freezer and put it on ice.
- The I15009 is placed in the ice.
- Take out the Soc from the -20°C freezer°C and leave in the incubater for 10 min
- Transfer the I15009 into eppendorf. Mix the DNA to the cells by vortex and leave in the ice for 5 min. Note= while doing this, flame should be on.
- The eppendord tube is placed in 42°C waterbath for 1 min exactly
- Add 250μL of Soc.
- The tube is then placed in the shaker for 1 hr
- Take 1 selective agar plate, Ampicillin (Why? Because the and leave it in the incubater upside down with lid open slightly to lit air through.
- Take out the tubes from the shaker
- Take out the plates from the incubater.
- Turn on the flame. Transfer the contents from each epppendorf tubes into each 4 plates.
- Add glass beads.
- Now take all 4 plates and shake for 8-20 sec
- Remove the glass beads
- The plates are then left overnight in incubater at 37°C.
Purification of B0034
Purification of I15009
Digestion of B0034
Digestion of I15009
pepare the gel 2%
run the gel
Phsically cut the gel of particular bands
Ligation of B0034 with I15009
- Add 7 μL of B0034
- Add 1μL of I15009
- Add 1μL of T4 ligase buffer
- Add 1μL of T4 ligase Total= 10μL
Transformation of ligated B0034_I15009
- Add 10μL of Ligated B0034_I15009 into competent cell, DH5α