944 pET30b-pfu-exo
Date:4/5/2018
[edit]PCR of 944 F1+F2
Template:367, cut out band then combine
PCR from 367 pEC-Pfu
1, fragment 1
944-Pfu-Exo-Nde1-F
atcatcCATATGATTTTAGATGTGGATTAC
944-Pfu-exo-R
cgtgatagagggttGctatTtcgaaggcaagaatc
Fragment 2
944-Pfu-exo-F
gattcttgccttcgaAatagCaaccctctatcacg
944-Pfu-Exo-Xho1-R
atcatcctcgagggattttttaatgttaagccag
(ctggcttaacattaaaaaatccctcgag gatgat)
2, purify and join by PCR
944-Pfu-Exo-Nde1-F
atcatcCATATGATTTTAGATGTGGATTAC
944-Pfu-Exo-Xho1-R
atcatcctcgagggattttttaatgttaagccag
(ctggcttaacattaaaaaatccctcgag gatgat)
3, digest with Nde1 -Xho1
ligate into Nde1-Xho1 digested pET30b
or ligate into pMiniT2 first