Premium Oligonucleotide Synthesis - Gene Link · Premium Oligonucleotide Synthesis 1 Gene Link l...
Transcript of Premium Oligonucleotide Synthesis - Gene Link · Premium Oligonucleotide Synthesis 1 Gene Link l...
P r e m i u m O l i go nuc l e o t i de S y n t he s i s
1 Gene Link l 1-800-GENE LINK l www.genelink.com
QUALITY • CONSISTENCY • CONFIDENCE
Gene Link oligos are for demanding applications
and consistent results. We believe that investigators who
value time and have no room for an experiment to fail due to
oligo quality should consider Gene Link. Our numerous
quality control steps for each oligo assure confidence.
An actual gel photo of each oligo
is affixed on the oligo report. An
absolute testimony of quality.
Gene Link has raised the standard
since inception over a decade ago.
We have the pictures to prove it!
Actual Gel Photo
G O L D STAN D A R D
G O L D STAN D A R D
Gene Link l 1-800-GENE LINK l www.genelink.com 2
The Gene Link Advantagen Stringent Quality Control
Measures
n Specializing in Long Oligos up to 250 mer
n Trityl Monitoring of All Oligos
n Polyacrylamide Gel Photograph of Each Oligo
n All Modifications Available
n All Oligo Types Available
n Easy Online Ordering System
n Online Design and Analysis Tools
n Knowledgeable Technical Support
n Personalized, Friendly Customer Service
250
100%
100%
100%
Oligo size
Yiel
dYi
eld
Yiel
d
Coupling efficiency 99.50%
Coupling efficiency 99.00%
Coupling efficiency 98.00%
Coupling Efficiency and Full Length Oligo Yield
Oligo Size 99.50% 99.00% 98.00%
20 90.916 82.617 68.12340 82.243 67.573 45.4860 74.398 55.268 30.36380 67.301 45.204 20.27
100 60.881 36.973 13.533120 55.074 30.24 9.034140 49.821 24.734 6.031160 45.068 20.23 4.027180 40.769 16.546 2.688200 36.88 13.533 1.795220 33.36 11.07 1.19240 30.18 9.05 0.8250 28.7 8.19 0.65
Superior to “Mass-Produced Factory Oligos”Gene Link is not an oligo factory.Each oligo is synthesized, processedand quality assured to Gene Link’sabsolute standards. This includescoupling efficiency monitoring ofeach base during synthesis and electrophoretic analysis of each oligo on a polyacrylamide gel to visually assess quality.
Coupling EfficiencyWe maintain a coupling efficiencythreshold of greater than 99.5% forall oligos by using premium reagentsof exacting specifications, membranesynthesis, state-of-the-art instrumentsand optimized software-driven protocols. This may not be evidentwhen comparing short oligos, asPCR and sequencing reactions arevery robust and can tolerate up to50% failure/truncated sequence oligos. However, you are clearly taking a chance by using long oligossynthesized at anything below99.5% coupling efficiency.
Trityl MonitoringAll Gene Link DNA synthesizers areequipped with trityl monitors formonitoring coupling efficiency ofeach added base. The instrumentsare programmed to halt when it fallsbelow the threshold.
See example of routine trityl bars.
Long Oligos
Ask our competitors how oftenthey synthesize 200 to 250 mer
oligonucleotides. Gene Link specializes in long oligos.
You are invited to compare.
Sequence length
Routine Trityl Coupling Efficiency
Actual trityl coupling efficiency of a 210 mer.
Oligo Design and Analysis Tools
3 Gene Link l 1-800-GENE LINK l www.genelink.com
Gene Link has been leading theway by providing the most userfriendly online experience inoligo ordering. From oligo designand analysis, to the convenientordering system and the assur-ance of a secured transaction,Gene Link provides the mostcomprehensive web resource inthe industry.
Features include convenientNCBI blasting and secondarystructure analysis, simple importtools for large orders in spread-sheet or text file format, andthree levels of review and editing.
Applications include RNAiExplorer™, a robust siRNAsearch and design tool, and astandalone Oligo Explorer™application for online acquisitionof sequences and oligo design.
Custom Oligo Ordering System• Classic ordering system with
extensive analysis features
• Timesaver multi oligo import fromspreadsheet and text files
• Ability to handle mixed oligo types,purity and modifications in a singleorder
• Selection of oligo type (DNA, RNA,Phosphorothioate, Chimeric, etc.)
• Simple drop down menu selectionfor 5', internal or 3' modifications
• Analyze for oligo hairpin and loops
• Integrates with NCBI Blast forhomology checks
• Flip 3' to 5' and reverse complement
Online Oligo Analysis• Simulate annealing, loops and
hairpin formations
• Calculate MW, EC, Tm, A260, etc.
Too busy to order all of your oligos in
one session? Gene Link’s answer to
the multitasking researcher with end-
less interruptions is the “Save Session“
feature. Enter as many oligos as you
wish, click the “Save Session“ button
and resume at your will. Your oligos
will be saved. What’s more, you'll save
money on shipping by consolidating
your multiple orders into one.
Save Session
C u s t o mO l i go nuc l e o t i de
S y n t he s i s
Gene Link l 1-800-GENE LINK l www.genelink.com 4
Multiple Oligo NCBI BlastClick to ascertainhomologies to other sequences.
Perform NCBI Blast of multiplesequences at once by using GeneLink’s online MultiBlast application.Import all the sequences using aspreadsheet or a text file. All of yoursequences will be blasted and resultsretrieved. Gene Link offers a veryconvenient approach to perform multiple blast searches.
Molecular BiologyConvenience AppletsGene Link has numerousonline applets for quick calculations. The BioCalculatoris a series of applets for sim-plifying the routine laboratory calculations. Thefollowing convenient calcula-tors are available:
• Oligo Resuspension
• Oligo Dilution
• Oligo Tm
• Reagent Dilution
• Molarity Determination
• Ligation
• Base/Dye Ratio
Oligo Explorer™A PC-based application for standaloneDNA sequence retrieval and oligo design.
Oligo Explorer™ was developed todesign PCR and sequencing primers.Oligo Explorer™ is an efficient easy-to-use tool to determine primer prop-erties like Tm, GC%, primer loops andprimer dimers.
Oligo Explorer™ also includes a pow-erful “Primer Wizard” tool that helpsyou to find suitable primer pairs. Youcan set your own parameters for theprimer pair search engine or use thedefault parameters. “Primer Wizard”suggests primer pairs that amplifyPCR products of the given length.Individual primer pairs are suggestedthat theoretically will not form stableprimer dimers or primer loops.
Oligo Specifications Report
Custom Oligo SpecificationsGene Link custom oligonucleotidesare supplied desalted and lyophilized.They are ready to use after appropri-ate reconstitution. Dry oligonu-cleotides are stable at roomtemperature for an extended periodof time.
Storage & ReconstitutionThe oligonucleotide should preferablybe frozen upon receipt. TE buffer (10 mM Tris, 1 mM EDTA, pH 7.5) isrecommended for dissolving theoligonucleotides. After reconstitutionstore the stock solution at –80°C or–20°C.
Purity & UsageThe crude, desalted oligonucleotidesupplied is suitable for all amplifica-tion and sequencing protocols. Gelpurification is advised for all oligosused for cloning applications and foroligos longer than 50 mer.
Biophysical DataEach oligo after desalting is quanti-fied by recording A260. Exact nmolsand µg are determined by the extinc-tion coefficient and molecular weightof the oligo.
Gel Photo DocumentationAn actual gel picture of the synthe-sized custom oligonucleotide is supplied. A major single band represents high purity of the crudeoligonucleotide.
5 Gene Link l 1-800-GENE LINK l www.genelink.com
Gene Link’s CustomOligonucleotide SynthesisReport specifies each oligoname and sequence alongwith its pertinent physicalproperties such as MW,%GC, Tm, A260 units, etc.Our report is also unique inthat we affix an actual poly-acrylamide gel electrophore-sis photograph onto eachreport, so that you also mayvisually attest to the qualityof our product.
From your custom oligo tothe presentation of our oligosynthesis report, not a stepof quality is overlooked. Youare invited to compare.
C u s t o mO l i go nuc l e o t i de
S y n t he s i s
Gene Link l 1-800-GENE LINK l www.genelink.com 6
Unmodified DNA Oligo Synthesis*
Scale of Synthesis Catalog No. Price ($)
50 nmol 26-6400-05 0.90
200 nmol 26-6400-02 2.00
1 µmol 26-6400-01 3.75
2 µmol 26-6400-03 6.50
10 µmol 26-6400-10 32.00
15 µmol 26-6400-15 38.00
*minimum charge for 15 mer applies. Please visit www.genelink.comfor current list prices. Call for institutional discount pricing structure.
Oligo Scale of Synthesis and Typical Yield
Crude Desalted RPC Purified** Gel Purified20 mer oligo* 30 mer oligo* 50 mer oligo*Typical yield Typical yield Typical yield
Scale A260 Units nmols mg A260 Units nmols mg A260 Units nmols mg
50 nmol 8-10 30+ 0.2-0.3 4-5 12+ 0.1-0.16 NR* [1-2] NR* [2-4] NR* [0.03-0.06]
200 nmol 20-25 80+ 0.6-0.8 8-12 24+ 0.26-0.4 4-6 8+ 0.13-0.2
1 µmol 100-120 400+ 3-4 40-50 30+ 1.3-1.6 20-25 40+ 0.6-0.8
Purity & Yield
*Yield of 30 µg/A260 unit for oligos is calculated for an ~equimolar base composition. Long stretches of a single base or homopolymers will have variable yields. Example for homopolymeric 50 mer: A(50) = ~20/A260 Unit; G(50) = ~28/A260 Unit; T(50) = ~35/A260 Unit and C(50) = ~39/A260 Unit.
Purity is greater than 80% depending on oligo sequence andstructure. Refer to coupling efficiencytable for oligo length dependent purityand yield.
No further purification required forPCR and sequencing applications.
Gel purification recommended for oli-gos above 50 mer and all applicationsinvolving cloning and mutagenesis.
Purity 85% to 95% depending on oligo sequence andstructure.
Yield and purity will be lower forsequences with high GC content.
Not recommended for oligos longerthan 35 mer.
**RPC is reverse phase purification using acartridge; a substitute for HPLC.
Purity 98% to ~100% depending on oligo sequence and struc-ture.
Yield will gradually decrease as length ofoligo increases. Palindromes, hairpins andhigh GC content oligos and oligos con-taining stretches of 3 or more G’s inducesstrong secondary structure and basestacking thus decreasing purity and yield.
NR* Not Recommended
Purification
Scale of Synthesis Price ($)/purification
Product Catalog No. 50 nmol 200 nmol 1 µmol 2 µmol 10 µmol 15 µmol
Gel Purification 26-6400-XX 75.00 75.00 150.00 280.00 1500.00 1800.00
Reverse Phase Cartridge 26-6400-XX 30.00 30.00 90.00 170.00 750.00 900.00
Design your oligos today and use them tomorrow
morning! Investigators who just can not wait order
our rush service (order by 12 noon EST). We ship the
same day for next early morning delivery in the US
and 72 hours for most international destinations.
* Turn-around time stated is for unmodified oligos.
Please inquire about purified and modified oligos
Same Day Oligo*
Gen
e Li
nk
14
0 O
ld S
aw M
ill
Riv
er R
oad
|H
awth
orn
e, N
Y 1
05
32
|1
-80
0-G
ENEL
INK
|te
l: 9
14
-76
9-1
19
2|
fax:
91
4-7
69
-11
93
|em
ail:
ord
ers@
gen
elin
k.co
m|
ww
w.g
enel
ink.
com
™
CUST
OM
OLI
GO
SP
ECIF
ICAT
ION
S
Qu
ali
ty•
Co
ns
iste
nc
y•
Co
nfi
de
nc
e
OLI
GO
SY
NTH
ES
IS
Mob
ility
of
an o
ligon
ucle
otid
e is
dep
ende
nt u
pon
the
size
and
bas
e co
mpo
sitio
n. O
ligos
of
the
sam
e si
ze m
ay n
ot s
hare
the
sam
e m
obili
ty p
atte
rns
base
d on
the
follo
win
g m
igra
tion
rate
C>A
>T>G
. A s
tret
ch G
's an
d GC
's in
duce
s st
rong
sec
onda
ry s
truc
ture
tha
t tr
avel
s as
hig
her m
obili
ty fr
agm
ents
.
Lane
Oli
goN
ame
Sequ
ence
(5'
–3')
Size
MW
TMnm
ols
µg
A26
0U
nits
1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
11.
Prim
er 1
Prim
er 2
Prim
er 3
Prim
er 4
Prim
er 5
Prim
er 6
Prim
er 7
Prim
er 8
Prim
er 9
Prim
er 1
0
Prim
er 1
1
CAT
CC
TGC
AGG
GC
TAG
CTC
ATAG
AGC
TTG
CG
CG
TCA
ATT
AGG
ATAC
CTA
GG
GG
TGC
TCTA
GAT
CAG
GAG
CTT
GC
GC
AGTC
CC
CG
TGG
GG
ATAC
CTA
GTC
ACG
TAC
TAC
TATG
TCA
CAT
CC
TGC
AGG
GC
TAG
CTC
ATAG
AGC
TTG
CG
CG
TCA
ATT
AGAG
CTT
GG
CTC
AAG
CAG
GA
AAT
CG
GG
AGC
GG
CAC
TTC
GTA
CG
GC
GC
GTC
C
CG
GA
ATTC
GG
TCAC
AGG
CTT
GG
TCA
GG
TCTG
TCTG
GG
ATC
CC
A
AAG
AGA
AAG
GTA
GG
AAG
CAC
CC
AAC
CTC
CTG
TCC
ACC
AAC
TTTC
TTTC
GTT
GG
ATG
TCC
ATC
TGC
GG
CG
TTTA
TGTT
GG
TTC
TCC
TGTA
GG
ACTG
GA
A
TGG
TCAG
AAT
TCTA
GC
CTT
TCG
TGAC
GA
AAT
TTTA
ACAT
AA
AAG
AA
AGG
CTT
CTT
GAT
ATAT
TATC
AAG
AA
ACC
TTTC
TTTT
CTA
TTA
AAT
TTAC
A
AAT
TCTC
AGTA
CTG
TGTT
TCAG
CAG
AAG
GAG
TCTT
ACAT
GTG
ATG
GG
GTG
TTAC
AAC
TGA
AA
AGTC
AA
AAG
AAG
TTTG
TATT
ACC
ATTT
TCA
ATAG
CAG
TATA
AA
AGG
TTC
TCTT
TG
GAT
TCC
AGTT
GTT
GC
TGC
TTTA
CTA
CTC
TTTC
TAG
TGC
TTAG
CT
CTC
AAG
CAG
GA
AAT
CG
AGC
GG
CAC
TTC
GTA
CG
TAA
ATG
CC
AA
51 64 48 42 25 18 20 78 96 161
42
15,7
15
19,7
19
14,8
00
12,9
50
7,69
8
5,50
7
6,25
7
23,8
69
29,5
13
49,7
36
12,9
25
74.8
77.9
74.6
77.0
63.9
56.6
53.4
77.7
70.2
76.2
71.1
48.7
47.8
45.5
48.9
48.2
52.6
40.2
9.3
8.5
6.2
28.6
765.
3
941.
9
672.
7
633.
6
370.
7
289.
8
251.
5
222.
7
252.
0
309.
4
369.
8
26.6
1
32.1
1
23.1
0
21.9
2
12.7
9
9.49
10.3
7
7.26
9.14
10.8
7
13.5
3
Cust
omer
Nam
e:
Alys
on R
odge
rs
Cust
omer
Num
ber:
10
532A
J1
Orde
r N
umbe
r: 1
3603
9
Date
: J
une
13,
2004
NO
TES
Olig
os 1
-7 a
re c
rude
unp
urifi
ed.O
ligos
8-1
1ar
e ge
l pur
ified
.Gel
lane
s fo
r ol
igos
8-1
1co
rres
pond
to c
rude
follo
wed
by
gel p
urifi
ed.
OLI
GO
SY
NTH
ES
IS
Cust
om O
ligo
Spe
cifi
cati
ons
Gene
Lin
k cu
stom
olig
onuc
leot
ides
are
sup
plie
d de
salt
ed a
nd ly
ophi
lized
. Th
ey a
rere
ady
to u
se a
fter
app
ropr
iate
rec
onst
itut
ion.
Dry
olig
onuc
leot
ides
are
sta
ble
at r
oom
tem
pera
ture
for
an
exte
nded
per
iod
of t
ime.
Hai
rpin
Str
uctu
res
One
esse
ntia
l ele
men
t of
eff
icie
nt p
rimer
des
ign
is t
o m
inim
ize
inte
rnal
sec
onda
ry s
truc
ture
, es
peci
ally
hai
rpin
loop
s w
hich
ten
dto
be
dece
ptiv
ely
stab
le a
t st
anda
rd a
nnea
ling
tem
pera
ture
s.Hai
rpin
s ar
e st
able
wit
h as
few
as
4 ba
ses
stac
ked
in t
he s
tem
and
a lo
op s
ize
of 4
to
6 ba
ses.
The
sta
bilit
y de
clin
es a
s th
e lo
op s
ize
incr
ease
s. T
he s
tem
and
loop
siz
e ar
e re
late
d pr
opor
tion
atel
y su
chth
at lo
nger
ste
m s
izes
can
tol
erat
e lo
nger
loop
siz
es (
4).
As a
gen
eral
rul
e, a
void
hai
rpin
s w
ith
mor
e th
an 3
bas
es i
n th
est
em.
Stab
le h
airp
in lo
op f
orm
atio
n dr
asti
cally
red
uces
the
prim
erco
ncen
trat
ion
avai
labl
e fo
r hy
brid
izat
ion
to t
he t
arge
t se
quen
ce.
Base
Com
posi
tion
Hig
her
GC c
onte
nt s
tabi
lizes
hyb
ridiz
atio
n, b
ut a
str
ing
of G
's an
dC'
s ca
n ex
hibi
t in
tern
al H
oogs
teen
bas
e pa
iring
, no
n-W
atso
n-Cr
ick
base
pai
ring
and
shou
ld b
e av
oide
d (3
,4).
Alt
houg
h th
is a
nom
-
alou
s be
havi
or i
s di
ffic
ult
to p
redi
ct,
thes
e st
ruct
ures
can
disr
upt
stab
le p
rimer
bin
ding
. In
gen
eral
, av
oid
runs
of
mor
e th
an t
hree
con
secu
tive
G's
in p
rimer
s. A
lso,
exa
m-
ine
pote
ntia
l prim
ers
for
self-
com
plem
enta
ry a
nd h
airp
inst
ruct
ures
. Nu
clea
r M
agne
tic
Reso
nanc
e (N
MR)
stu
dies
have
sho
wn
that
a s
tabl
e ha
irpin
can
for
m w
ith
just
fou
rG-
C ba
sepa
irs i
n th
e st
em a
nd j
ust
thre
e ba
ses
in t
helo
op (
5).
Refe
renc
es1.
Mic
hael
Zuk
er (
2003
) Nu
clei
c Ac
ids
Res.
,31
, 34
06–3
415.
2. S
anta
Luci
a, J
. (1
998)
Pro
c. N
at.
Acad
. Sc
i. US
A 95
, 14
60.
3. S
aroc
hi,
M-T
., Co
urto
is,
Y.,
Gusc
hlba
uer,
W.
1970
. Eu
r. J.
Bio
chem
.14
: 41
1.4.
Gen
e Li
nk,
Inc.
int
erna
l dat
a.5.
Sum
mer
, M
.F.,
Byrd
R.A
., Ga
llo,
K.A.
, Sa
mso
n, C
.J.,
Zon,
G.,
Egan
, W
. 19
85.
Nucl
eic
Acid
s Re
s.13
: 63
75.
R26-
6400
-XXB
100
6200
4
©Ge
ne L
ink,
Inc
. 20
04 A
ll Ri
ghts
Res
erve
d
PR
OD
UCT
GU
IDE
Oli
go S
cale
of
Synt
hesi
s an
d Ty
pica
l Yi
eld
of U
nmod
ifie
d O
ligo
s*
Crud
e De
salt
edRP
C Pu
rifi
ed**
*Ge
l Pur
ifie
d20
mer
olig
o**
30m
er o
ligo*
*50
mer
olig
o**
Scal
eA
260
Uni
tsnm
ols
A26
0U
nits
nmol
sA
260
Uni
tsnm
ols
50 n
mol
8-10
30+
4-5
12+
NR*
[1-2
]NR
*[2
-4]
200
nmol
20-2
5 80
+8-
12
24+
4-6
8+
1 µm
ol10
0-12
040
0+40
-50
30+
20-2
540
+
Puri
ty&
Yiel
d
*The
yie
ld o
f m
odifi
ed o
ligos
var
ies
base
d on
mod
ifica
tion
.
**Yi
eld
of 3
0µg
/A26
0un
it f
or o
ligos
is
calc
ulat
ed f
or a
n ~e
quim
olar
bas
e co
mpo
siti
on.
Long
str
etch
es o
f a
sing
le b
ase
or h
omop
olym
ers
will
hav
e va
riabl
e yi
elds
.Ex
ampl
e fo
r ho
mop
olym
eric
50
mer
: A
(50)
=~2
0/A 2
60Un
it;
G(50
)=~2
8/A 2
60Un
it;
T(50
)=~3
5/A 2
60Un
it a
nd C
(50)
=~3
9/A 2
60Un
it.
*** R
PC i
s re
vers
e ph
ase
purif
icat
ion
usin
g a
cart
ridge
; a
subs
titu
te f
or H
PLC.
NR*
Not
Reco
mm
ende
d.Purit
y is
mor
e th
an 8
0% d
epen
ding
on o
ligo
sequ
ence
and
str
uctu
re.
Purit
y 85
% t
o 95
% d
epen
ding
on
olig
ose
quen
ce a
nd s
truc
ture
. No
t re
com
men
ded
for
olig
os lo
nger
tha
n 35
mer
.
Purit
y 98
% t
o ~1
00%
dep
endi
ng o
n ol
igo
sequ
ence
and
str
uctu
re.
Yiel
d w
ill g
radu
ally
decr
ease
as
leng
th o
f ol
igo
incr
ease
s.
Gen
e Li
nk™
Hai
rpin
Loo
p Fo
rmat
ion
and
Prim
er D
esig
n*
Seq
uenc
e5'
-CA
GC
GC
AC
TA
CA
GG
CAT
GA
CG
T-3'
5'-G
TC
CG
CA
CG
TA
CG
GA
CAT
-3'
5'-G
TC
AG
CC
GC
AC
GTA
CG
GA
CAT
-3'
5'-A
GTA
AC
GC
AC
TAC
GG
AC
TTA
CG
AC
-3'
22m
er;
dG=
-47.
5; T
m(N
N)=
61.6
°C18
mer
; dG
=-3
8.4;
Tm
(NN)
:57.
0°C
21m
er;
dG:-
46.3
; Tm
(NN)
:61.
70°C
24m
er;
dG=
-47.
1; T
m(N
N)=
58.8
°C
* Dim
ers
5' C
AG
CG
CA
CT
AC
AG
GC
AT
GA
CG
T3'
5' G
TC
CG
CA
CG
TAC
GG
AC
AT3'
5' G
TC
AG
CC
GC
AC
GTA
CG
GA
CAT
3'5'
AG
TA
AC
GC
AC
TAC
GG
AC
TT
AC
GA
C 3
'|
||
|+
||
||
||
++
||
||
||
+|
| |
|+
++
++
++
+3'
TG
CA
GTA
CG
GA
CA
TC
AC
GC
GA
C 5
'3'
TA
CA
GG
CAT
GC
AC
GC
CT
G 5
'3'
TA
CA
GG
CA
TG
CA
CG
CC
GA
CT
G 5
'
3' C
AG
CA
TT
CA
GG
CA
TC
AC
GC
AA
TG
A5'
STAC
K AT
3 I
S 4
BP L
ONG.
STAC
K AT
8 I
S 6
BP L
ONG.
STAC
K AT
11
IS 6
BP
LONG
.ST
ACK
AT 2
IS
4 BP
LON
G.dG
=-4
.8;
Tm=
-58.
4°C
dG=
-5.7
; Tm
=-4
2.4°
CdG
=-4
.65;
Tm
=-2
8.2°
CdG
=-2.
05;
Tm=-
47.3
°C
Hai
rpin
None
5'G
TC
CG
CA
C5'
GT
CA
GC
CG
CA
C5'
AG
TA
AC
GC
AC
TLo
ops
||
||
|]
| |
|]
||
||
]3'
TAC
AG
GC
ATG
3'TA
CA
GG
CA
TG
3' C
AG
CA
TT
CA
GG
CA
STEM
AT
1 IS
5 B
P LO
NG.
LOOP
=6.
STEM
AT
6 IS
3 B
P LO
NG.
LOOP
=6
STEM
AT
2 IS
4 B
P LO
NG.
LOOP
=12
.dG
=-5
.3;
Tm=
87.3
°CdG
=-2
.4;
Tm=
68.9
°CdG
=0.
8; T
m=
13.8
°C
*Sec
onda
ry s
truc
ture
res
ults
are
tru
ncat
ed t
o sh
ow t
he m
ost
stab
le s
truc
ture
s. A
ll th
erm
odyn
amic
val
ues
incl
udin
g Tm
and
sec
onda
ry s
truc
ture
s ca
lcul
ated
and
dis
play
ed s
olel
y in
dica
te t
he r
elat
ive
stab
ility
of
the
seco
ndar
y st
ruct
ures
. Th
ey s
houl
d on
ly b
e us
ed t
o co
mpa
re t
he r
elat
ive
stab
ility
of
the
stru
ctur
es.
dG v
alue
uni
t is
kca
l/m
ol.
Visi
t w
ww.
gene
link.
com
/too
ls/g
l-SO
D.as
p to
des
ign
olig
os o
r cl
ick
on t
he ‘A
naly
ze’ b
utto
n w
hile
on
the
onlin
e ol
igo
orde
ring
pag
e.
Stor
age
& Re
cons
titu
tion
Th
e ol
igon
ucle
otid
e sh
ould
pre
fera
bly
befr
ozen
upo
n re
ceip
t. T
E bu
ffer
(10
mM
Tris
,1
mM
EDT
A, p
H 7
.5)
is r
ecom
men
ded
for
diss
olvi
ng t
he o
ligon
ucle
otid
es.
Afte
rre
cons
titu
tion
sto
re t
he s
tock
sol
utio
n at
-80
°C o
r -2
0°C.
Gel
Phot
o Do
cum
enta
tion
An a
ctua
l gel
pic
ture
of
the
synt
hesi
zed
cust
om o
ligon
ucle
otid
e is
sup
plie
d. A
maj
or s
ingl
e ba
nd r
epre
sent
s hi
gh p
urit
yof
the
cru
de o
ligon
ucle
otid
e.
Puri
ty &
Usa
geTh
e cr
ude,
des
alte
d ol
igon
ucle
otid
e su
pplie
d is
sui
tabl
e fo
r al
l am
plif
icat
ion
and
sequ
enci
ng p
roto
cols
. Ge
l pur
ific
atio
nis
adv
ised
for
all
olig
os u
sed
for
clon
ing
appl
icat
ions
and
for
olig
os lo
nger
tha
n50
mer
.
Biop
hysi
cal
Data
Each
olig
o af
ter
desa
ltin
g is
qua
ntif
ied
byre
cord
ing
A 260
. Ex
act
nmol
s an
d µg
is
dete
rmin
ed b
y th
e ex
tinc
tion
coe
ffic
ient
an
d m
olec
ular
wei
ght
of t
he o
ligo.
Succ
essf
ul u
se o
f ol
igos
as
prim
ers
for
ampl
ifica
tion
and
sequ
enci
ng s
tart
s w
ith
func
tiona
l prim
er d
esig
n fo
llow
ed b
y op
tim
ized
PCR
am
plifi
catio
n co
ndit
ions
.Fo
rtun
atel
y, b
oth
PCR
and
sequ
enci
ng r
eact
ions
are
in
here
ntly
‘rob
ust’
and
have
bee
n ob
serv
ed t
o to
lera
te w
ide
varia
tions
in q
ualit
y of
prim
ers
whe
n us
ing
uniq
ue t
em-
plat
es.
The
sam
e ‘to
lera
nce’
can
als
o le
ad t
o fa
lse
prim
ing,
poor
res
ults
and
fru
stra
ting
tim
e lo
ss w
ith
tem
plat
es o
fhi
gher
com
plex
ity.
Prim
er s
peci
ficit
y al
one
does
not
gua
rant
ee a
n op
tim
umam
plifi
cati
on y
ield
. Nu
mer
ous
com
pute
r ap
plic
atio
ns a
reav
aila
ble
for
prim
er s
earc
h an
d de
sign
. M
ost
of t
hese
appl
icat
ions
do
not
cons
ider
the
eff
ect
of h
airp
in s
truc
-tu
res
whi
ch t
end
to b
e qu
ite
stab
le t
herm
odyn
amic
ally
.Ge
nera
l gui
delin
es f
or p
rimer
des
ign
are
give
n be
low
fol
-lo
wed
by
a br
ief
acco
unt
of s
tabl
e ha
irpin
str
uctu
re
form
atio
n an
d no
n-W
atso
n-Cr
ick
base
pai
ring
indu
ced
by
a st
retc
h of
G’s
and
G’s
inte
rspe
rsed
wit
h A’
s or
C’s
(1-3
).
Gene
ral G
uide
lines
1. S
peci
fici
ty:
Sele
ct a
n 18
to
24m
er s
tret
ch w
ith
perf
ect
spec
ifici
ty.
2. B
ase
Com
posi
tion
:Pr
efer
ably
mai
ntai
n GC
con
tent
bel
ow60
% w
ith
no s
tret
ches
of
mor
e th
an 3
G’s
or 4
run
s of
the
sam
e ba
se.
3. T
m:
Sele
ct p
rimer
Tm
wit
hin
a fe
w d
egre
es o
f th
e pa
ir.
4. C
ross
Hom
olog
ies:
Perf
orm
NCB
I bl
ast
to d
eter
min
eex
tent
of
cros
s ho
mol
ogie
s.
5. S
econ
dary
Str
uctu
re:
Perf
orm
com
pute
r as
sist
ed a
naly
sis
to v
iew
for
mat
ion
of s
tabl
e di
mer
s, lo
ops
and
hairp
ins.
Prim
er D
esig
n