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Bacterial
Expression System >
Expresso ® T7
產品特色
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Ready-to-use
vectors and competent cells - NO ligation step.
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High
efficiency: ›90% recombinants.
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6XHis-N / -C
selection.
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Save time!
Avoid days of vector and cell preparation time.
說明書下載
Datasheet
Kit
Contains
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pETite N/C-His
Expression Vector,
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Positive
Control Insert DNA
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DNA primers
for screening inserts by PCR and sequencing --- T7 F/R
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HI-Control 10G
Chemically Competent Cells, and HI-Control
BL21(DE3)Chemically Competent Cells.
產品包裝:
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5 Reactions |
10 Reactions |
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ExpressoTM
T7 Cloning and Expression System, N/C-His Combo |
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49000-1 |
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ExpressoTM
T7 Cloning and Expression System, N-His |
49001-1 |
49001-2 |
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ExpressoTM T7 Cloning and Expression System, C-His |
49002-1 |
49002-2 |
pETite™ Vectors
RBS, ribosome binding site; ATG, translation start site; Stop,
translation end site; Kan, kanamycin resistance gene; ROP, Repressor
of Priming (for low copy number); Ori, origin of replication.
CloneSmart® transcription terminators (T) prevent transcription into
or out of the insert, and a T7 terminator follows the cloning site.
The 6xHis affinity tag is fused to the amino terminus (pETite N-His)
or at the carboxyl terminus (pETite C-His) of the expressed protein
of the target protein.
High cloning efficiency with HI-Control
10G Chemically Competent cells
High cloning efficiency with Expresso T7 System. Pre-processed
pETite C-His vector was mixed with 1 µl of unpurified PCR gene
product and transformed into HI-Control 10G Chemically Competent
Cells. Colony PCR was performed on 18 randomly chosen colonies; 17
of 18 contained insert of the correct size.
pETite vectors protein production
The pETite vectors have low copy
number, similar to that of pBR322 plasmids
(~20 copies/cell), yielding 0.5–1.0
μg of plasmid DNA per ml of culture.
Primer design for target gene
amplification
Ex: Fusion to an N-terminal 6xHis
tag (pETite N-His Kan vector):
Forward primer (defined vector sequence includes 6 His codons):
5’-CAT CAT CAC CAC CAT CAC XXX2 XXX3 XXX4 XXX5 XXX6 XXX7 XXX8
(XXX2-XXX8 represents codons 2 through 8 of the target coding
region).
Reverse primer (defined vector sequence includes Stop anticodon):
5’-GTG GCG GCC GCT CTA TTA XXXn XXXn-1 XXXn-2 XXXn-3 XXXn-4
XXXn-5 XXXn-6 (XXXn - XXXn-6 represents the sequence
complementary to the last 7 codons of the target coding region).
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pETite C-His Kan vector: Forward primer
設計注意在XXX之前要加入ATG，而reverse則不需加TTA! |
Expression of various
proteins using the Expresso T7 System versus a pET Vector
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1,3,5,7,9,11: NO IPTG induced.
2,4,6,8,10,12: IPTG (1mM) induced 3 hours
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Purification of Active Soluble
Fluorescent Protein (YFP) Using 6xHis Tag
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