Gene specific primers:

GSS-F:
SbfI Kozak GSS
AGGCT cctgcagg CCACCATG NNNNNNNNNNNNNNNNNNNNN

GSS-R:
GSS * Xho1
(NNNNNNNNNNNNNNNNNNNNN TAG ctcgag ACCCAGCTTTC)

For example: GAAAGCTGGGT ctcgag CTA N(15–25)

 

Universal Primers

attL1-F
ccccGATGAGCAATGCTTTTTTATAATGCCAACTTTGTACAAAAAAGCA GGCTcctgcaggCCACCATG

attL2-R
(3’TAG ctcgag ACCCAGCTTTCTTGTACAAAGTTGGCATTATAAGAAAGCATTGCTTATCcccc5′)
ggggGATAAGCAATGCTTTCTTATAATGCCAACTTTGTACAAGAAAGCTGGGTctcgagCTA

 

1st PCR

2.5 ul of 10 X Pfu PCR buffer
0.5 ul of dNTPs (10 mM)
1 ul of cDNA or gDNA (~20 ng/ml)
0.5 ul of GSS-F (10 uM)
0.5 ul of GSS-R (10 uM)
0.2 ul of Pfu (5 U/ul)
sterile water to 25 ul

95 oC for 2 min
95 oC for 30 s
55 oC for 30 s
68 oC for 6 min (1 to 1.5 min per kb insert)
15 cycles
72 oC for 5 min

 

2nd PCR

5 ul of 10 X Pfu PCR buffer
1 ul of dNTPs (10 mM)
x ul pf 1 round PCR product
1 ul of attL1-F (10 uM)
1 ul of attL2-R (10 uM)
0.4 ul of Pfu (5 U/ul)
sterile water to 50 ul

95 oC for 2 min
95 oC for 30 s
55 oC for 30 s
68 oC for 6 min (1 to 1.5 min per kb insert)
15 cycles
72 oC for 5 min

 

Gel Purify

Optional

 

LR reaction

4 ul of LR Reaction buffer (5x)
3–8 ul of the 2nd PCR product (50–300 ng)
0.5 ul of destination vector (100–250 ng)
4 ul of Gateway LR Clonase mix
Final reaction volume: 20 ul
Incubate O/N at room temperature