5) Let cool at room temperature. 6) For ligation mix: - 1 µL of 10X buffer. - 2 µL of 5X PEG8000 solution. - 10-15 ng of vector. - 1 µL of ligase. - H2O up to 10 µL.
1) In a 1.5 mL Eppendorf tube, mix: - 2 µL of each oligonucleotide (100 µM) - 2 µL of 10X denaturation Buffer - 14 µL ddH20 2) Seal the tube with parafilm and place it in a Becher full of tap water 3) Heat the water until it starts to boil 4) Remove it from the microwave and keep the tube immerged. 5) Let cool at room temperature 6) For ligation mix: -
1 µL of 10X buffer
-
2 µL of 5X PEG8000 solution
-
10-15 ng of vector
-
1 µL of ligase
-
H2O up to 10 µL
7) Incubate at room temperature for 2 hours 8) Transform 2 µL of ligation
Denaturation Buffer (10X) 500 mM Tris-HCl pH = 7.4 100 mM MgCl2
Experiments have been done showing that after 2 hours 95% of the product obtain after overnight ligation was obtained. Molecular ratio calculation: vector insert.
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