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BamHI 5’ G-G-A-T-C-C
C-C-T-A-G-G 5’
“STAR” ACTIVITY
Altered or relaxed specificity
cleaving sequences similar (not identical) to their
defined recognition sequence
RE’s with known star activity:
Apo I , Ase I , BamH I, BssH II, EcoR I, EcoR V, Hind III,
Hinf I, Pst I, Pvu II, Sal I, Sca I, Taq I, Xmn I
Caused by:
High units to µg of DNA ratio [Varies with each enzyme,
usually >100 units/µg]
Low ionic strength [<25 mM]
High pH [>pH 8.0]
Presence of organic solvents [DMSO, ethanol, ethylene
glycol, dimethylacetamide, dimethylformamide,
sulphalane]
Substitution of Mg++ with other divalent cations [Mn++,
Cu++, Co++, Zn++]
DIGESTION WITH MULTIPLE REs
If with compatible buffers: Digest with
both enzymes in the same buffer.
If with slightly incompatible buffers:
Cut with one enzyme, then alter the
buffer composition and cut with the
second enzyme.
If with totally incompatible buffers:
Perform one digestion, recover the DNA
(usually by precipitation) and
resuspend in the buffer appropriate for
the second enzyme.
RESULTS
4 bio 2
4 bio 3
4 bio 4
4 bio 5
4 bio 6
Expression of Recombinant Blo t 11-fD in Escherichia
coli
Clone 1A
Clone 1B
Clone 2A
Clone 2B
Clone 1B
Clone 2B
Clone 1A
Clone 2A
5000bp
marker
5000bp
marker
3000 3000
1000
1000
750 666bp
750 666bp 500
500 250
250
PCR products
RE digested Plasmids using BamHI and XhoI
Verification
of fD gene insert
Plasmid pGEX-4T-1