PCR Protocol (50µl)

Buffer Pfu 5 µl
Upper primers 2.5 µl
Lower primers 2.5 µl
MgCL2 1 µl
DNA (depends on concentration, usually 0.5µl)
Enzime Taq Polymerase 1 µl
DNTP’s 8 µl
Water 29.5 µl

Cycles :)
95ºC 4 min
95ºC 1 min
55ºC :30 secs
70ºC 1 min
goto 2:4 30 times
72ºC 5 min

Hot start PCR protocol

Preheated 105ºC
Heated Lid ON
Pause OFF
In denat 94ºC 5 min.
Hot start OFF

30 cycles
94 ºC 30 secs.
55ºC 30 secs.
70ºC 45 secs.
72ºC 5 min
hold 10ºC

Ligation

These are done in 1 mL eppendorf tubes and normally have a T.V. of 20 µl.

• Add A µl of the insert (plasmid digested with corresponding enzymes).
• Add B µl of vector (plasmid digested with corresponding enzymes and complementary so that the ligation can be done properly) and properly DEPHOSPOHATED.
• Add 2 µl T4 DNA ligase buffer.
• Add 20-A-B-3-1 µl H2O miliQ.
• Add 1 µl T4 DNA ligase enzyme.

Transformation

• Pipette the plasmid to a 1.3 mL eppendorf with competent cells and mix with the pipette.
• Incubate tubes on ice for 42 minutes.
• Heatshock at 42ºC for 2 minutes.
• Incubate for 5 minutes on ice.
• Add 1 mL LB broth to each tube.
• Mix by inversion 2-3 times.
• Incubate at 37ºC for an hour with shaking.
• During this hour prepare LB agar plates (30 mL) containing the appropriate antibiotics.
• After the hour Plate 100 µl.
• Centrifuge at 10000 rpm for 2 minutes.
• Drain 700-800 µl of supernatant, mix pellet with the rest and plate. Add to the plate.
• Let grow overnight at 37ºC.

Liquid culture

• Once the bacteria has grown, add 4 mL LB broth to a glass tube.
• Add the appropriate antibiotic.
• With a wooden toothpick take one colony per tube. Place the toothpick in the tube and vortex.
• Incubate the tube in the shaker 37ºC for at least 6 hours.