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QuikChange XL Site-Directed
Mutagenesis Kit
INSTRUCTION MANUAL
Catalog #200516 (30 reactions) and #200517 (10 reactions)
Revision C
For In Vitro Use Only
200516-12
L
IMITED
P
RODUCT
W
ARRANTY
This warranty limits our liability to replacement of this product. No other warranties of any kind,
express or implied, including without limitation, implied warranties of merchantability or fitness for
a particular purpose, are provided by Agilent. Agilent shall have no liability for any direct, indirect,
consequential, or incidental damages arising out of the use, the results of use, or the inability to use
this product.
O
RDERING
I
NFORMATION AND
T
ECHNICAL
S
ERVICES
United States and Canada
Agilent Technologies
Stratagene Products Division
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La Jolla, CA 92037
Telephone
(858) 373-6300
Order Toll Free
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Location
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United Kingdom
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0800 917 3282
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Technical Services
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00800 7400 7400
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00800 7400 7400
0800 919 289
00800 7400 7400
0800 182 8234
00800 7400 7400
0800 023 0448
00800 7400 7400
0800 563 081
00800 7400 7400
0800 917 3281
All Other Countries
Please contact your local distributor. A complete list of distributors is available at
www.stratagene.com.
QuikChange XL Site-Directed Mutagenesis Kit
C
ONTENTS
Materials Provided.............................................................................................................................. 1
Storage Conditions .............................................................................................................................. 1
Additional Materials Required .......................................................................................................... 1
Notice to purchaser ............................................................................................................................. 2
Introduction......................................................................................................................................... 3
QuikChange XL Mutagenesis Control.............................................................................................. 5
Mutagenic Primer Design................................................................................................................... 6
Primer Design Guidelines...................................................................................................... 6
Additional Primer Considerations ......................................................................................... 7
XL10-Gold Ultracompetent Cells ...................................................................................................... 8
Protocol ................................................................................................................................................ 9
QuikSolution ......................................................................................................................... 9
Mutant Strand Synthesis Reaction (Thermal Cycling).......................................................... 9
Dpn
I Digestion of the Amplification Products................................................................... 11
Transformation of XL10-Gold Ultracompetent Cells ......................................................... 11
Transformation Guidelines .............................................................................................................. 14
Storage Conditions .............................................................................................................. 14
Aliquoting Cells .................................................................................................................. 14
Use of 14-ml BD Falcon Polypropylene Round-Bottom Tubes.......................................... 14
Use of
β-Mercaptoethanol...................................................................................................
14
Quantity of DNA Added ..................................................................................................... 14
Length and Temperature of the Heat Pulse ......................................................................... 14
Preparing the Agar Plates for Color Screening ................................................................... 14
Troubleshooting ................................................................................................................................ 15
Preparation of Media and Reagents ................................................................................................ 16
References .......................................................................................................................................... 17
Endnotes............................................................................................................................................. 17
MSDS Information............................................................................................................................ 17
Quick-Reference Protocol ................................................................................................................ 20
QuikChange XL Site-Directed Mutagenesis Kit
M
ATERIALS
P
ROVIDED
Quantity
Materials provided
PfuTurbo
DNA polymerase (2.5 U/μl)
10× reaction buffer
c
Dpn
I restriction enzyme (10 U/μl)
Oligonucleotide control primer #1 [34-mer (100 ng/μl)]
5´ CCA TGA TTA CGC CAA GCG CGC AAT TAA CCC TCA C 3´
Oligonucleotide control primer #2 [34-mer (100 ng/μl)]
5´ GTG AGG GTT AAT TGC GCG CTT GGC GTA ATC ATG G 3´
pWhitescript 4.5-kb control plasmid (5 ng/
μl)
QuikSolution
dNTP mix
d,e
XL10-Gold ultracompetent cells
f
(yellow tubes)
XL10-Gold
β-mercaptoethanol
mix (β-ME)
pUC18 control plasmid (0.1 ng/μl in TE buffer
c
)
a
Catalog #200516
a
30 reactions
80 U
500
μl
300 U
750 ng
750 ng
50 ng
500
μl
30
μl
10 × 135
μl
2 × 50
μl
10
μl
Catalog #200517
b
10 reactions
25 U
500
μl
100 U
750 ng
750 ng
50 ng
500
μl
10
μl
4 × 135
μl
50
μl
10
μl
b
c
d
e
f
The QuikChange XL Site-Directed Mutagenesis Kit (Catalog #200516) contains enough reagents for 30 total reactions,
which includes 5 control reactions.
The QuikChange XL Site-Directed Mutagenesis Kit (Catalog #200517) contains enough reagents for 10 total reactions,
which includes 5 control reactions.
See Preparation
of Media and Reagents.
Thaw the dNTP mix once, prepare single-use aliquots, and store the aliquots at –20°C.
Do not subject the dNTP mix
to multiple freeze-thaw cycles.
The composition of the dNTP mix is proprietary.
This reagent has been optimized for the QuikChange XL site-
directed mutagenesis protocols and has been qualified for use in conjunction with the other kit components. Do not
substitute with dNTP mixes provided with other Stratagene kits.
Genotype: Tet
r
Δ
(mcrA)183
Δ
(mcrCB-hsdSMR-mrr)173
endA1 supE44 thi-1 recA1 gyrA96 relA1 lac
Hte
[F’
proAB lacI
q
Z
Δ
M15
Tn10 (Tet
r
) Amy Cam
r
]
S
TORAGE
C
ONDITIONS
XL10-Gold Ultracompetent cells, XL10-Gold
β-ME,
and pUC18 Control Plasmid:
–80°C
All Other Components:
–20°C
A
DDITIONAL
M
ATERIALS
R
EQUIRED
14-ml BD Falcon polypropylene round-bottom tubes (BD Biosciences Catalog #352059)
5-Bromo-4-chloro-3-indloyl-β-
D
-galactopyranoside (X-gal)
Isopropyl-1-thio-β-
D
-galactopyranoside (IPTG)
Revision C
© Agilent Technologies, Inc. 2009.
QuikChange XL Site-Directed Mutagenesis Kit
1

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