Pierre Lindenbaum (lindenb at jouy.inra.fr) wrote:
: ------------------------------------------------------------------------------
: SubCloning
: ..............................................................................
: CloneIt V1.0 has found a solution:
: Digest VECTOR with EcoRI [G^AATTC] (878) and Sal I [g^tcgac] (894).
: 5' --G.CCG.G/AA.TT C.CCG.GGG.ATC.CG/T.CGA. CCT.GCA.GCC.AAG-- 3'
: 3' --C.GGC.C TT.AA/G.GGC.CCC.TAG.GC A.GCT./GGA.CGT.CGG.TTC-- 5'
: NH2 P E F P G I R R P A A K .COOH
: Digest first with EcoRI .Then treat with Klenow DNA polymerase.Finally digest
: with Sal I .
: Digest INSERT with Sca I [AGT^ACT] (1034) and Sal I [g^tcgac] (3605).
: 5' --AT.AAA.GT/ A.CTT.TCA.AAG.AAA.G-- --TA.GAG./TCG.A CC.TGC.AGG.CAT.G-- 3'
: 3' --TA.TTT.CA/ T.GAA.AGT.TTC.TTT.C-- --AT.CTC. AGC.T/GG.ACG.TCC.GTA.C-- 3'
: NH2 K V L S K K E -- -- E S T C R H A .COOH
: Treat with T4 DNA polymerase.
: Sites wil be in frame ligated in 5'.
: The first stop codon detected BEFORE the EcoRI site (878) is localized at
: position 428 on vector. The first stop codon detected AFTER the Sca I
: site (1034) is localized at position 3558 on insert.
: Digestion post-ligation: no enzyme was found.
