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.