Download Cloning a Gene for Over-expression and Purification

Cloning a gene
Cloning a Gene for Over-expression and Purification
Motivation: produce a bioremediation enzyme to
metabolize herbicide residues in soil.
Nitroreductase is able to metabolize herbicides such as 2,4dinitrophenol. However this enzyme denatures readily: is it too delicate
for field work.
NADH-oxidase (NADOX) is a homologous enzyme from Thermus
thermophilus that has a very high melting temperature and at 25° C
displays increased catalytic activity in the presence of denaturants and
high salt concentrations.
We want to over-express NADOX and test if for possible use as a
bioremediation tool.
Strategy: clone the gene into an overexpression
plasmid.
A.-F. Miller © 2013 Page 1
Simplest example
Cloning vectors contain an
origin of replication, a
selectable marker, cloning
sites.
Splicing a gene of interest
into a plasmid provides for
its propagation by host cells
and permits its ready
repurification (retrieval).
A.-F. Miller, 2013, pg
3
G&G Fig. 12.1
A clone is a (population of) genetically identical organisms.
In order for the gene to be carried over multiple
generations, it needs to be replicated at least as often as
the cell in which it is carried. For convenience, we clone
bacterial genes into plasmids.
Plasmid, small extrachromosomal circle of DNA. (4.3 kB vs.
4.6 MB) Includes origin of replication. Often carries a set of
genes needed for metabolism of occasional nutrient sources,
or antibiotics.
A.-F. Miller, 2013, pg
Cloning operations do
not depend on the
content of the gene.
Restriction enzymes
vector, insert.
sticky ends / blunt ends
Phosphatase, ligase
Directional cloning behind a promotor,
transformation of ‘competent’ cells,
selection.
A.-F. Miller, 2013, pg
4
G&G Fig. 12.3, 5
Making lots of the insert: use PCR
PCR amplification
PCR: Polymerase chain reaction is used to make many copies
of DNA lying between the primers used.
Primers are necessary in order for DNA polymerase to copy
template DNA.
Template
Polymerase
5’ to 3’
A.-F. Miller, 2013, pg
Primer
Supply template, primers (two) NTP, DNA polymerase that
tolerates repeated heating (to separate DNA strands) and
cooling (to permit primers to anneal onto template).
A.-F. Miller, 2013, pg
5
6
PCR see also Fig 12.21
PCR, the video
+
+
+
+
melt 95°C
cool 70°C
+
linkers bind
+NTP
polymerase
primers at
x1000
+
+
+
+
+
+
+
+
+
+
+
+
+
A.-F. Miller, 2013, pg
7
+
+
+
+
+
+
+
+
A.-F. Miller, 2013, pg
8
Also http://www.youtube.com/watch?v=x5yPkxCLads
DNA Replication
Restriction endonucleases
!
!
!
!
!
!
Restriction enzymes are enzymes that cut DNA at specific sequences
within double stranded DNA.
Different enzymes cut DNA at different sequences.
Target sequences are usually palindromic (read the same in both
directions).
These enzymes can be used to confirm the presence of sequences by
virtue of their action of cutting DNA only when the sequence occurs.
These enzymes often cut the two DNA strands in staggered locations,
and so are also used to produce complementary overhangs “sticky ends”.
Sticky ends allow different digestion products to be assembled together
to produce recombinant DNA molecules from two pieces that have been
produced by the same restriction enzymes.
We will be using Nde I (CATATG) and Hind III (AAGCTT)
TATGGCTTGGAAT3’
ACCGAACCTTA5’
Nde I
5‘CGTACCGATCA
3‘GCATGGCTAGTAT
5‘CGTACCGATCATATGTACCGGAT3’
5‘CGTACCGATCATATGGCTTGGAAT3’
+
3‘GCATGGCTAGTATACTGGAATA5’
3‘GCATGGCTAGTAT ACCGAACCTTA5’
TATGTACCGGAT3’
ACATGGAATA5’
A.-F. Miller, 2013, pg
9
http://www.wehi.edu.au/education/wehitv/
A.-F. Miller, 2013, pg
Over-expression vectors
pET plasmid provides a
strong promotor and other
‘designer’ features in the
service of expressing a
gene of interest.
10
Our Choice:
Vector: pET15TEV_NESG
EcoRI (5740)
AatII (5669)*
gene of interest
AmpR
PstI (4992)*
Provides
N-terminal His tag,
TEV cleavage site
ribosome binding site,
(but no lac repressor).
A.-F. Miller © 2013 Page 11
We choose to use a DNA
polymerase that has no
other duties in the cell, and
that we can up-regulate at
will (induce) upon addition
of IPTG to the medium.
(Look up IPTG)
rbs
SphI (725)*
BsaI (4808)*
pBR322 origin
Insert gene here
NheI (32)*
EcoRV (192)
T7 term
T7 reverse
XhoI (328)*
AccI (344)
SacI (354)*
BamHI (359)*
EcoRI (368)
NdeI (377)*
TEV
His
NcoI (423)*
XbaI (462)*
T7
T7 forward
BglII (528)*
pET15TEV_NESG
5741 bp
ApaI (1461)*
BssHII (1661)*
EcoRV (1700)
BsmBI (1865)
AccI (3624)
BsmBI (3495)
Init
Sal I
XhoI
Nco I|
Nde I
EcoR I
BamH I
Sac I"
HinD III
|
|
TEV Protease
|
|
|
|
|
|
|
CCATGGGCCATCACCATCACCATCACgaaaacctgtattttcagagcCATATGGCGAATTCTGCGGATCCTGCGAGCTCTGTCGACGCAAAGCTTCTCGAG
GGTACCCGGTAGTGGTAGTGGTAGTGcttttggacataaaagtctcgGTATACCGCTTAAGACGCCTAGGACGCTCGAGACAGCTGCGTTTCGAAGAGCTC
______6XHis tag____
A.-F. Miller © 2013 Page 12
( signals carried within a cloned insert. It therefore lacks
translation
the ribosome
PflM
I(572) binding site and
gin start codon present on the pET translation vectors. A C-terminalApaB
His•Tag I(674)
sequence is availori ATG
able. Unique sites are shown on the circle map. Note that the sequence is numbered by the pBR322
f1
convention, so the T7 expression region is reversed on the circular map. The cloning/expression
3153
3914-4771
4903-5358
®
Sca I(4464)
Pvu I(4354)
Sty I(57)
Bpu1102 I(80)
Pst pET-21(+)
I(4229)sequence landmarks
Vector: pET21
237-253
236
158-203
140-157
26-72
640-1719
3153
3914-4771
4903-5358
pET-21(+)
Dra III(5127)
Sca I(4464)
Pvu I(4354)
AlwN I(3507)
or
Bsa I(4045)
i (3Eam1105 I(3984)–
15
3)
(5369bp)
Mlu I(990)
Bcl I(1004)
BstE II(1171)
Bmg I(1199)
Apa I(1201)
BssH II(1401)
EcoR V(1440)
Hpa I(1496)
Psp5 II(2097)
Bpu10 I(2197)
or
)
Psp5 II(2097)
Bpu10 I(2197)
BspE I(2617)
gene
ATG
TAA
T7 promoter primer #69348-3
T7 promoter primer #69348-3
T7 promoter
lac operator
lac operator
BamH I EcoR I Sac I
BamH I EcoR I
Sac I
Eag
I
III 1102 Not
I
Sal I Hind Bpu
I
Bpu1102 I
Sty I
Sal I Hind III
Ava I*
Xho Sty
I I
Eag I
Not I
Ava I*
Xho I
His•Tag
His•Tag
T7 terminator
T7 terminator
T7 terminator primer #69337-3
T7 terminator primer #69337-3
pET-21(+) cloning/expression region
Novagen • ORDERING 800-526-7319 •
A.-F. Miller © 2013 Page 13
TECHNICAL SUPPORT
TECHNICAL SUPPORT
Init
Sal I
XhoI
Nco I|
Nde I
EcoR I
BamH I
Sac I"
HinD III
|
|
TEV Protease
|
|
|
|
|
|
|
CCATGGGCCATCACCATCACCATCACgaaaacctgtattttcagagcCATATGGCGAATTCTGCGGATCCTGCGAGCTCTGTCGACGCAAAGCTTCTCGAG
mcs of pET15TEV
Before proposing to use Nde I, Eco RI, Hind III or Xho I,
confirm that these do not cut within the gene.
Search for CATATG, GAATTC, AAGCTT, CTCGAG
NADOX gene
Gene sequence TTHA0425
NCBI Reference Sequence: NC_006461.1
>gi|55979969:402495-403112 Thermus thermophilus HB8 chromosome,
ATGGAAGCGACCCTTCCCGTTTTGGACGCGAAGACGGCGGCCCTAAAGAGGCGTTCCATCCGGCGTTACC
GGAAGGACCCCGTACCCGAGGGGCTTCTCCGGGAAATCCTCGAGGCCGCCCTCCGGGCGCCCTCGGCCTG
GAACCTCCAGCCCTGGCGGATCGTGGTGGTGCGGGACCCCGCCACCAAACGGGCCCTGAGGGAGGCGGCC
TTCGGCCAGGCCCACGTGGAGGAGGCCCCCGTGGTCCTGGTCCTCTACGCCGACCTCGAGGACGCTCTCG
CCCACCTGGACGAGGTCATCCACCCCGGGGTCCAGGGGGAAAGGCGTGAGGCGCAGAAGCAGGCCATCCA
ACGGGCCTTCGCCGCCATGGGGCAAGAGGCGCGAAAGGCCTGGGCCTCCGGGCAGAGCTACATCCTCTTG
GGCTACCTCCTTCTCCTCCTGGAGGCTTATGGCCTCGGAAGCGTCCCCATGCTGGGGTTTGACCCCGAGA
GGGTGAGGGCGATCCTGGGGCTTCCTTCCCACGCCGCCATCCCCGCCCTGGTGGCCTTGGGCTACCCGGC
GGAGGAGGGCTACCCCTCCCACCGCCTGCCCCTGGAGCGGGTGGTCCTCTGGCGCTAA
Cannot use Xho I
800-207-0144
A.-F. Miller © 2013 Page 14
pET-21(+) cloning/expression region
Novagen • ORDERING 800-526-7319 •
Eenie Meenie Minie Moe, which nuclease should we use ?
PshA I(1835)
i
(3 1
BspE
53 I(2617)
BspLU11 I(3091)
Sap I(2975)
Bst1107 I(2862)
Tth111 I(2836)
rbs
BssH II(1401)
EcoR V(1440)
Hpa I(1496)
PshA I(1835)
pET-21(+)
BspLU11 I(3091)
Sap I(2975) AlwN I(3507)
Bst1107 I(2862)
Tth111 I(2836)
T7 promoter
3-5358)
(490
gin
ori
0-1719)
I (64
lac
Provides
lac operator repress
expression of toxic gene,
C-terminal His tag.
Pst I(4229)
BstE II(1171)
Bmg I(1199)
Apa I(1201)
Bgl II(268)
SgrA I(309)
Sph I(465)
EcoN I(525)
PflM I(572)
ApaB I(674)
(5369bp)
f1
Ava I(158)
Xho I(158)
Eag I(166)
Not I(166)
Hind III(173)
Sal I(179)
Sac I(190)
EcoR I(192)
BamH I(198)
Ap (39
1447
71
)
T7 promoter
T7 transcription start
Multiple cloning sites
mH I - Xho I)
Bsa(BIa(4045)
His•Tag® coding sequence
T7 terminator
Eam1105 I(3984)
–
lacI coding sequence
pBR322 origin
bla coding sequence
f1 origin
Bcl I(1004)
0-1719)
I (64
lac
Second Choice:
region of the coding strand transcribed by T7 RNA polymerase is shown below. The f1 origin is
oriented so that infection with helper phage will produce virions containing single-stranded DNA
that corresponds to the coding strand. Therefore, single-stranded sequencing should be performed
Mlu I(990)
using the T7 terminator primer (Cat. No. 69337-3).
Ap (39
1447
71
)
pBR322 origin
bla coding sequence
f1 origin
Blue coloured codons
are corrections I made
based on literature
disagreements and the
crystal structure.
800-207-0144
Vector: pET15TEV_NESG
Design feature for the insert (our gene)
Sites we will use
Init
Sal I
XhoI
Nco I|
Nde I
EcoR I
BamH I
Sac I"
HinD III
|
|
TEV Protease
|
|
|
|
|
|
|
CCATGGGCCATCACCATCACCATCACgaaaacctgtattttcagagcCATATGGCGAATTCTGCGGATCCTGCGAGCTCTGTCGACGCAAAGCTTCTCGAG
GGTACCCGGTAGTGGTAGTGGTAGTGcttttggacataaaagtctcgGTATACCGCTTAAGACGCCTAGGACGCTCGAGACAGCTGCGTTTCGAAGAGCTC
______6XHis tag____
Add a linker between protease cleavage and our protein.
ATGGGCCATCACCATCACCATCACgaaaacctgtattttcagagcCATATGGCGAATTCTGCGGATCCTGCGAGCTCTGTCGACGCAAAGCTTCTCG
http://web.expasy.org/translate/
What codons to use for Gly and Ser ?
GS
5'3' Frame 1
Met G H H H H H H E N L Y F Q S H Met A N S A D P A S S V D A K L L
Therefore we will need the following flanking sequences on our
primers
‘5-CATATG➝
←TTCGAA-5’
Both of these endonucleases need some bases on either side in order to cut
efficiently, so we will add a few more bases.
Intelligent choice of codons
Insert a mini-linker of flexible soluble
amino acids.
Gly-SerTherefore add codons for Gly and Ser
to gene.
5’-TTTTTTCATATG➝
←TTCGAATTTTTT-5’
A.-F. Miller © 2013 Page 15
http://muta-tion.blogspot.com/2011/07/genetic.html
A.-F. Miller © 2013 Page 16
For Gly, use GGC
Hershberg R, Petrov DA (2009)
General Rules for Optimal Codon Choice.
PLoS Genet 5(7): e1000556. doi:10.1371/journal.pgen.1000556
For Ser, AGC ,
also Welch et al (2009)
Welch M, Govindarajan S, Ness JE, Villalobos A, Gurney
A, et al. (2009) Design Parameters to Control Synthetic
Gene Expression in Escherichia coli. PLoS ONE 4(9):
e7002. doi:10.1371/journal.pone.0007002
Note that just choosing the most common
codon does not guarantee higher expression.
Arg
Leu
Ser
Ile
A.-F. Miller © 2013 Page 17
A.-F. Miller © 2013 Page 18
Modified early sequence of our gene.
5’-TTTTTTCATATGGGCAGC➝
extension, restriction site, codons for linker
The authentic gene (early bases, corrections in blue, start in green)
ATGGAAGCGACCCTTCCCGTTTTGGACGCGAAGACGGCGGCCCTAAAGAGGCG
Meld together to get: (only first 30 bases to be replicated are shown)
5’-TTTTTTCATATGGGCAGCGAAGCGACCC TTCCCGTTTT GGACGCGAAG➝
How far into the gene does the primer need to extend ?
Long enough that it binds selectively (short sequences can recur elsewhere in the
genome simply at random), short enough that the search for
complementary sequence does not happen too slowly (fast binding and
release from wrong sites)
A.-F. Miller © 2013 Page 19
.
Hence the following “Rules of thumb” for primer design
5’ end of the gene
Restriction site permits splicing into vector. (we have the Nde I site)
ATG encodes start of translation. ( √)
follows a ribosome binding site and a transcription initiation site. (plasmid
provides these).
Extend the primer by 2-10 bases upstream of restriction site (√).
Overlap with the desired gene for enough bases to give Tm ≈ 60 °C (72°
preferred by some).
We will deal with these next.
GC content between 40-60 %.
Length of 18-30 bases for specificity.
NOT last three bases = G or C, not last base = T.
Tm = 2°C * (A + T) + 4°C * (C + G)
http://www.basic.northwestern.edu/biotools/oligocalc.html
SeeMiller
http://www.embl.de/pepcore/pepcore_services/cloning/pcr_strategy/primer_design/
A.-F.
© 2013 Page 20
Use tools on web sites to determine the GC content and melting
temperatures (Tm) of duplexes predicted from different lengths of
oligos.
Considering first round only when only black bases pair.
Might RNA structure mask the start site ?
5’ end of the gene
5’-TTTTTTCATATGGGCAGCGAAGCGACCC TTCCCGTTTT GGACGCGAAG➝
5’-TTTTTTCATATGGGCAGCGAAGCGACCC TTCCCGTTTT G➝
Range of Tm values account
for range of Na+
concentrations that may be
present (stabilizing duplex).
√ Predicted stability of -6.2 kcal/mol
(would like to have < 10).
Considering later rounds when daughter molecules from early
rounds are serving as templates.
http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi
5’-TTTTTTCATATGGGCAGCGAAGCGACCC TTCCCGTTTT GGACGCGAAG➝
49 % GC, Tm = 68 - 78 °C 39 bases
50 % GC, Tm = 67 - 74 °C 32 nucleotides
50 % GC, Tm = 69 - 79 °C 40 nucleotides
A.-F. Miller © 2013 Page 21
Only 21 b in rounds 1,2, 57 % GC,
Tm = 56 - 63 °C
But only 14 b long in round 1, 2,
64% GC, Tm 43-47 °C
22 bases long in round 1, 2, 59% GC, Tm 59-67 °C.
3’ end of the gene
Restriction site permits splicing into vector.
TAA stop codon (TAG and TGA are more prone to read-through).
Extend the primer by 2-10 bases upstream of restriction site.
Overlap with the desired gene for enough bases to give Tm ≈ 60 °C (72°
preferred by some).
GC content between 40-60 %.
Length of 18-30 bases for specificity.
NOT last three bases = G or C, not last base = T.
Check that the two primers do not pair with one another. Mutate to a better
stop codon. Add
Hind III site and
CAC CGC CTG CCC CTG GAG CGG GTG GTC CTC TGG CGC TAA AAGCTT AAAAAA 3’
extension.
CAC CGC CTG CCC CTG GAG CGG GTG GTC CTC TGG CGC TAG
5’
3’
The primer is the complement strand
3’ AC GGG GAC CTC GCC CAC CAG GAG ACC GCG ATT TTCGAA TTTTTT
The primer (written right way ‘round, with site of Hind III cut)
5’
5’ TTTTTT AAGCTT TTA GCG CCA GAG GAC CAC CCG CTC CAG GGG CA 3’
A.-F. Miller © 2013 Page 23
A.-F. Miller © 2013 Page 22
Reverse primer, Rules of thumb
3’ end of the gene
Reverse primer, Rules of thumb
5’
Vector
Init
Sal I
XhoI
Nco I|
Nde I
EcoR I
BamH I
Sac I"
HinD III
|
|
TEV Protease
|
|
|
|
|
|
|
CCATGGGCCATCACCATCACCATCACgaaaacctgtattttcagagcCATATGGGCAGCGCGAATTCTGCGGATCCTGCGAGCTCTGTCGACGCAAAGCTTCTCGAG
Restriction site permits splicing into vector. (√)
TAA stop codon (TAG and TGA are more prone to read-through). (√)
Extend the primer by 2-10 bases upstream of restriction site. (√)
Overlap with the desired gene for enough bases to give Tm ≈ 60 °C (72°
preferred by some).
GC content between 40-60 %.
Length of 18-30 bases for specificity.
NOT last three bases = G or C, not last base = T.
Check that the two primers do not pair with one another.
The primer (bases that pair in the first round are in bold,
all pair in rounds >2)
5’ TTTTTT AAGCTT TTA GCG CCA GAG GAC CAC CCG CTC CAG G 3’
50 % GC Tm 67-77 °C 36 bases long (23 bases in rounds 1,2, 70 % GC and Tm 64-72 °C)
45 % GC Tm 62-71 °C 31 bases long (18 bases in rounds 1,2, 67 % GC and Tm 55-61 °C)
Use green primer and a few extra cycles
A.-F. Miller © 2013 Page 24
(
gin
ori
f1
3153
3914-4771
4903-5358
Sca I(4464)
Pvu I(4354)
NEXT: pET21
I(572)
ATG start codon present on the pET translation vectors.PflM
A C-terminal
His•Tag® sequence is availApaB isI(674)
able. Unique sites are shown on the circle map. Note that the sequence
numbered by the pBR322
convention, so the T7 expression region is reversed on the circular map. The cloning/expression
region of the coding strand transcribed by T7 RNA polymerase is shown below. The f1 origin is
oriented so that infection with helper phage will produce virions containing single-stranded DNA
that corresponds to the coding strand. Therefore, single-stranded sequencing should be performed
using the T7 terminator primer (Cat. No. 69337-3).
Sty I(57)
Bpu1102 I(80)
pET-21(+) sequence landmarks
Forward
Forward primer
T7 transcription start
Multiple cloning sites
(BamH I - Xho I)
His•Tag® coding sequence
Bsa I(4045)
T7 terminator
Eam1105 I(3984)la–cI coding sequence
pBR322 origin
bla coding sequence
f1 origin
237-253
236
158-203
140-157
26-72
640-1719
3153
3914-4771
4903-5358
Dra III(5127)
f1
Sca I(4464)
49 % GC, Tm = 68 - 78 °C 39 bases Only 21 b in rounds 1,2, 57 % GC, Tm = 56 - 63 °C
Pvu I(4354)
Pst I(4229)
provide Eco RI, rbs and atg.
(NdeI underlined)
Bsa I(4045)
AlwN I(3507)
5’ TTTTTT AAGCTT TTA GCG CCA GAG GAC CAC C➝ 3’
45 % GC Tm 62-71 °C 31 bases long (18 bases in rounds 1,2, 67 % GC and Tm 55-61 °C)
Temperatures match well.
3-5358)
(490
gin
ori
(5369bp)
–
BssH II(1401)
EcoR V(1440)
Hpa I(1496)
PshA I(1835)
Reverse primer: as for pET15TEV
Psp5 II(2097)
Bpu10 I(2197)
Sap I(2975)
Bst1107 I(2862)
Tth111 I(2836)
BspE I(2617)
gene
ATG
BstE II(1171)
Bmg I(1199)
Apa I(1201)
Psp5 II(2097)
Bpu10 I(2197)
BspLU11 I(3091)
AlwN I(3507)
or
i (3
Sap I(2975)
15
3)
BspE I(2617)
Bst1107 I(2862)
Tth111 I(2836)
BspLU11 I(3091)
rbs
Mlu I(990)
Bcl I(1004)
PshA I(1835)
pET-21(+)
or
Eam1105 I(3984)
i 3
GAATTCGAAGGAGATATACAT ATG GAA GCG ACC CTT CCC GTT (TTG
15 GAC G...
3)
Check to be sure that your two primers will not bind strongly to one-another.
http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/
BssH II
EcoR V
Hpa I(1496)
0-1719)
I (64
lac
5’-TTTTTTCATATGGGCAGCGAAGCGACCC TTCCCGTTTT G➝
Reverse
Bgl II(268)
SgrA I(309)
Sph I(465)
EcoN I(525)
(1401)
PflM I(572)
ApaB I(674) (1440)
pET-21(+)
(5369bp)
BstE II(1171)
Bmg I(1199)
Apa I(1201)
Ap (39
1447
71
)
Primers for use with pET15TEV_NESG
Pst I(4229)
T7 promoter
Mlu I(990)
Bcl I(1004)
Ava I(158)
Xho I(158)
Eag I(166)
Not I(166)
Hind III(173)
Sal I(179)
Sac I(190)
EcoR I(192)
BamH I(198)
0-1719)
I (64
lac
Ap (39
1447
71
)
pBR322 origin
bla coding sequence
f1 origin
TAA
T7 promoter primer #69348-3
Consequences for expression:
At N-terminus, after TEV cleavage, we will add H M G S to the
‘front’ of the protein.
At the C-terminus there will be no changes relative to the native
protein.
A.-F. Miller © 2013 Page 25
T7 promoter
T7 promoter primer #69348-3
T7 promoter
lac operator
BamH I EcoR I Sac I
(rules of thumb)
Restriction site permits splicing into vector. (we have the Eco RI site)
ATG encodes start of translation. ( √)
Follows a ribosome binding site and a transcription initiation site. (√).
Extend the primer by 2-10 bases upstream of restriction site.
Overlap with the desired gene for enough bases to give Tm ≈ 60 °C
(72° preferred by some).
GC content between 40-60 %.
Length of 18-30 bases for specificity.
NOT last three bases = G or C, not last base = T.
provide Eco RI, rbs and atg. (NdeI underlined)
5’-TTTTGAATTCGAAGGAGATATACAT ATG GAA GCG ACC CTT CCC GTT T...
42 bases 40% GC 66-76 °C Tm in later rounds; 17 bases 59% GC 50-55 °C Tm in rounds 1,2
41 bases 39% GC 65-75 °C Tm in later rounds; 16 bases 56% GC 46-51 °C Tm in rounds 1,2
45 bases 42% GC 67-78 °C Tm in later rounds; 20 bases 60% GC 56-63 °C Tm in rounds 1,2
Use blue primer
A.-F. Miller © 2013 Page 27
BamH I EcoR I Sac I
Sal I Hind III
Bpu1102 I
Sty I
Eag
I
Bpu1102 I
Not I
Sal I Hind III
Ava I*
Xho I
Sty I
Eag I
Not I
Ava I*
Xho I
His•Tag
T7 terminator
His•Tag
T7 terminator primer #69337-3
T7 terminator
pET-21(+) cloning/expression region
T7 terminator primer #69337-3
Novagen • ORDERING 800-526-7319 •
A.-F. Miller © 2013 Page 26
TECHNICAL SUPPORT
800-207-0144
pET-21(+) cloning/expression region
Novagen • ORDERING 800-526-7319 •
Forward primer:
lac operator
TECHNICAL SUPPORT
800-207-0144
Primers for use with pET21
Forward
5’ TTTTGAATTCGAAGGAGATATACAT ATG GAA GCG ACC CTT CCC GT➝
45 bases 42% GC 67-78 °C Tm in later rounds; 20 bases 60% GC 56-63 °C Tm in rounds 1,2
Reverse
5’ TTTTTT AAGCTT TTA GCG CCA GAG GAC CAC C ➝3’
31 bases 45 % GC Tm 62-71 °C (18 bases in rounds 1,2, 67 % GC and Tm 55-61 °C)
Temperatures match alright.
Check to be sure that your two primers will not bind strongly to one-another.
http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/
Consequences for expression:
At N-terminus, The native protein will be intact (no changes).
At the C-terminus there will be no changes relative to the native
protein.
A.-F. Miller © 2013 Page 28