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[ANNOUNCE]CloneIt :Finding Cloning Strategies

Pierre Lindenbaum lindenb at jouy.inra.fr
Wed Oct 21 03:09:15 EST 1998


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.




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