1. Gently mix the following reaction by pipetting and centrifuge briefly.

　 　 　 　 　 　 　 　 　 　 　 　 　 　 　

	20 μl system	50 μl system
Template	12～20 ng	30～50 ng
Forward primer	1.0 μl	2.5 μl
Reverse primer	1.0 μl	2.5 μl
dNTP	1.6 μl	4.0 μl
10x Buffer	2.0 μl	5.0 μl

Program of KOD DNA polymerase

　 　 　 　 　 　 　 　 　 　 　 　 　 　 　 　 　

Temperature	Time
94 ℃	3 min.	25～30 cycles
94 ℃ (Denaturation)	40 sec
57.5 ℃ (Annealing)	30 sec
72 ℃ (Extension)	Depend on sequence size (2 kbp/min. for Taq)
72 ℃	5 min.
4 ℃	∞

2. Confirm the size of the digested product by gel electrophoresis.

3. Gel purification of the target size.

1. Digestion (vector)

　 　 　 　 　 　 　 　 　 　 　 　 　 　 　

Plasmid	200 ng	1000 ng
EcoRI / SpeI	0.2 μl	1 μl
XbaI / PstI	0.2 μl	1 μl
CutSmart Buffer	2 μl	5 μl
ddH2O	Up to 20 μl	Up to 50 μl
Digestion at 37℃ for 2.5hr.

2. Digestion (insert)

　 　 　 　 　 　 　 　 　 　 　 　 　 　 　

Plasmid	200 ng	1000 ng
EcoRI / XbaI	0.2 μl	1 μl
SpeI / PstI	0.2 μl	1 μl
CutSmart Buffer	2 μl	5 μl
ddH2O	Up to 20 μl	Up to 50 μl
Digestion at 37℃ for 2.5hr.

3.Confirm the size of the digested product by gel electrophoresis.

4. Gel purification of the target size.

5. Ligation

　 　 　 　 　 　 　

Vector (2 kbp)	molar ratio = 1:3 (can up to 1:10 depending on the DNA sizes)
Insert (1.5 kbp)
Quick Ligase Reaction Buffer (2X)*	10 μl
Quick Ligase	1 μl
ddH2O	Up to 20 μl

6. Transform the product by heat shock.

Gel Dissociation 1. Gel Extraction a. Excised the DNA fragment from the agarose gel. b. Transferred up to 300 mg of the gel slice to a 1.5 ml microcentrifuge tube. c. Added 500 μl of the Gel/PCR Bufffer to the sample and mixed by vortex. Incubate at 55~60℃ for 10 minutes (or until the gel slice has completely dissolved). d. During the incubation, mixed by vortexing the tube every 2~3 minutes. e. Cooled the dissolved sample mixture to the room temperature. DNA Binding 2. Placed a PG Column in a Collection Tube. Apply the supernatant to the PG Column by decanting or pipetting. 3. Centrifuged at 16,000 xg for 30 seconds. 4. Discarded the flow-through and place the PG Column back into the same collection tube. Wash 6. Added 400 μl of the Buffer W1 into the PG Column. 7. Centrifuged at 16,000 xg for 30 seconds. 8. Discarded the flow-through and place the PG Column back into the same collection tube. 9. Added 600 μl of the Buffer W2 (ethanol added) into the PG Column. 10. Centrifuged at 16,000 xg for 30 seconds. 11. Discarded the flow-through and place the PG Column back into the same collection tube. 12. Centrifuged at 16,000 xg again for 2 minutes to remove the residual Buffer W2. Elution 13. To elute the DNA, placed the PG Column in a clean 1.5 ml microcentrifuge tube. 14. Added 50 μl of the H2O (pH is between 7.0 and 8.5) to the center of each PG Column, let it stand for at least 2 minutes, and centrifuge at 16,000 xg for 2 min.