Biomedical Engineering Reference
In-Depth Information
a
pET28-TevH
NcoI His Tag SacII KpnI BamHI
CC ATG G
GC AGC AGC CAT CAT CAT CAT CAT CAC T
CC GCG G
GT GAA AAC CTG TAC TTC CAG
GGT ACC
ATT
GGA TCC
GAA TTC GAG CTC
GG TAC CCG TCG TCG GTA GTA GTA GTA GTA GTG AGG CGC CCA CTT TTG GAC ATG AAG GTC CCA TGG TAA CCT AGG CTT AAG CTC GAG
M G S S
H H H H H H
S A G
E N L Y F Q G
T I G S E F E L
His Tag TEV recognition Site
CCA TG
G
e
HindIII
CGT GAC
AAG CTT
GCG GCC GCA CTC GAG CAC CAC CAC CA
GCA CTG TTC GAA CGC CGG CGT GAG CTC GTG GTG GTG GT
GAC AAG
CTG TTC
b
pET28-IFN
α
8
TPIFN8_30oF
NcoI His Tag
CC ATG G
GC AGC AGC CAT CAT CAT CAT CAT CAC
NcoI
His Tag
CC ATG G
CC ATG GGC AGC AGC CAT CAT CAT CAT CAT CAC
GC AGC AGC CAT CAT CAT CAT CAT CAC
TCC GCG GGT GAA AAC CTG TAC TTC CAG GGT
TCC GCG GGT GAA AAC CTG TAC TTC CAG GGT
C
TGT GAT CTG CCT CAG ACT CAC AG
----
GG TAC CCG TCG TCG GTA GTA GTA GTA GTA GTG AGG CGC CCA CTT TTG GAC ATG AAG GTC CCA ACA CTA GAC GGA GTC TGA GTG TC----
M G S S
H H H H H H
S A G
E N L Y F Q G
C D L P Q T H
His Tag TEV recognition Site
IFN
α
8 gene
HindIII
-----------CAA AAA AGA TTG AAG AGT AAG GAA TAG
AAG CTT
GCG GCC GCA CTC GAG CAC CAC CAC CA
-----------
GTT TTT TCT AA
C TTC TCA TTC CTT ATC
TTC GAA CGC CGG CG
T GAG CTC GTG GTG GTG
GT
TPIFN8_30olR
Fig.
2
Cloning of the IFN
8 gene into the recipient vector pET28-TevH by the Transfer-PCR (TPCR) reaction.
(
a
) Sequence of the expression cassette in the pET28-TevH. The cassette includes N-terminal 6xHis-tag, TEV
protease recognition site (sequences are
underlined
and
marked
), and multiple cloning sites (some of the
restriction enzyme sites are indicated and
underlined
).
Arrows
indicate the planned integration sites of the
IFN
α
8 gene into the expression cassette. (
b
) Sequence of the expression cassette following integration of
the IFN
α
8 are shown. Primer names and directions are
shown. Forward (TPIFN8_30olF) and reverse (TPIFN8_30olR) primers include recipient vector sequences
(marked in
blue color
) and IFN
α
8 gene. Only the N- and C-terminal sequences of IFN
α
α
8-specifi c sequences (marked in
red color
).
Asterisk
indicates the translation
stop codon (TAG) of the IFN
α
8 gene (color fi gure online)
3. Synthetic primers (forward and reverse) for TPCR cloning.
Both primers are diluted with TE buffer (10 mM Tris-HCl,
1 mM EDTA, pH 8) or sterile double-distilled water, to 1
M
stock solution (
see
Fig.
2
,
Note 2
, and Subheading
3.1
for
primer design).
4. Plasmid DNA purifi cation kit (QIAprep spin mini prep kit,
Qiagen, Hilden, Germany; Catalog # 27106) or equivalent.
5. Donor and recipient plasmids. Both plasmids are diluted with
TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8) or sterile
double-distilled water, to 10 ng/
μ
L stock solution.
6. Spectrophotometer (Nano-Drop/Thermo Fisher Scientifi c,
Wilmington, DE; Catalog # ND-1000) or equivalent.
7. Thermocycler machine (SensoQuest, Goettingen, Germany;
Labcycler, Triple Block 3x21).
8. DpnI (New England Biolabs, Ipswich, MA; Catalog # R0176S)
or equivalent.
μ
