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Site-directed Mutagenesis Kit
CAT. NO.: mB005-S, mB005-L
Package: 10 rxn, 20 rxn
Kit Contents
Component
mB005-S，10 rxn mB005-L，20 rxn
10x Pfu Buffer with MgSO4
200 µl
400 µl
5x GC-rich Buffer with MgSO4
200 µl
400 µl
25 mM MgSO4 Solution
200 µl
400 µl
10 mM dNTP Mix
20 µl
40 µl
Pfu DNA Polymerase, 2.5 U/µl
15 µl
30 µl
Dpn I, 10 U/µl
12 µl
24 µl
pUC19 White Control Plasmid,5ng/µl 20 µl
40 µl
Control Forward Primer, 10 µM
15 µl
30 µl
Control Reverse Primer, 10 µM
15 µl
30 µl
Sterilized ddH2O
1 ml
2 ml
Protocol
1
1
Note
DH5α Competent Cells is not supplied in this kit.
Storage and stability
Please store at –20°C. When stored properly, the kit is stable till see package.
Introduction
The site-directed mutagenesis kit is used to make point mutations, replace nucleotides, and delete or insert single or
multiple adjacent nucleotides. The kit allows a fast, simple, and highly efficient introduction of specific mutations, insertions
and/or deletions of up to 12 nucleotides into a double-stranded plasmid of up to 8 kb. The entire procedure, including
transformation, may be completed in less than 3 hours.
Two complementary mutagenic primers with centrally located mutation sites are required to generate an expected
mutation site. The two primers anneal to opposite strands on the target vector and Pfu DNA Polymerase is then used to
amplify the entire target vector. The PCR amplification generates large numbers of mutated plasmids with staggered nicks.
Then use Dpn I restriction endonuclease to digest the residual methylated template and any hemi-methylated DNA. The
nicked vector DNA with the desired mutation site is transformed into competent cell DH5α, which can repair the nicked
vector.
The 2.68 kb pUC19 While Control Plasmid contains a frame-shifting point deletions of “AG” within the Hind III
Updated:07/2014
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restriction site sequences of lacZα gene. The mutant control plasmid shows white colonies on plates containing
X-galactosidase (X-gal). The control plasmid also confers ampicillin resistance; therefore, for selection, grow the E. coli on
plates containing ampicillin (and X-gal). In addition, the pUC19 While Control Plasmid can’t be digested by Hind III while its
reverse mutation can, however be digested by Hind III as in the control experiment.
Standard protocol
1.
The principles of mutagenic primer design
a.
The mutagenic primer set must contain the expected mutation and anneal to opposite strands on the target vector.
b.
Primers should be 30-45 bases with the melting temperature (Tm) of 75°C to 85°C.
c.
The expected mutation (replace, deletion or insertion) can be in the middle of the primer with ~11–21 bases of correct
sequence on both sides.
d.
Optimal primers should have a minimum GC content of 40% and terminate with G or C.
e.
For more details about the mutagenic primer design, Please refer to the PrimerX, a free online tool for automated
design of mutagenic primers for site-directed mutagenesis. The overlapping mutagenic primers designed by PrimerX
are also applied for the kit.
2.
Mutagenesis PCR
a.
Prepare the control reaction as indicated below
Component
Volume
10x Pfu Buffer with MgSO4
5 µl
10 mM dNTP Mix
1 µl
Control Forward Primer, 10 µM
1 µl
Control reverse Primer, 10 µM
1 µl
pUC19 White Control Plasmid, 5ng/µl
1 µl
Pfu DNA Polymerase, 2.5 U/µl
1.2 µl
Sterilized ddH2O
39.8 µl
b.
Prepare the sample reaction(s) as indicated below
Component
Volume
10xPfu Buffer with MgSO4
5 µl
10 mM dNTP Mix
1 µl
Forward Primer, 10 µM
1 µl
Reverse Primer, 10 µM
1 µl
Template Plasmid, 10 ng/µl
0.5~1 µl
Pfu DNA Polymerase, 2.5 U/µl
1.2 µl
Sterilized ddH2O
to 50 µl
Updated:07/2014
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c.
If the template plasmid is GC-rich, please prepare the sample reaction(s) as indicated below
Component
Volume
5xGC-rich Buffer with MgSO4
10 µl
10 mM dNTP Mix
1 µl
Forward Primer, 10 µM
1 µl
Reverse Primer, 10 µM
1 µl
Template Plasmid, 10 ng/µl
0.5-1 µl
Pfu DNA Polymerase, 2.5 U/µl
1.2 µl
Sterilized ddH2O
to 50 µl
d.
Place the samples in a cycler and start PCR by the following cycling program
Step
Temperature, °C Time
Number of Cycles
Initial Denaturation
95
3 min
1
Denaturation
95
30 s
Annealing
60
1 min
Extension
68
2 min/kb
Final Extension
68
10 min
1
Hold
4
N.A.
N.A
3.
12-18
Dpn I Digestion of the Amplification Products
Add 1 μl of the Dpn I restriction enzyme (10 U/μl) directly to each amplification reaction. Gently and thoroughly mix each
reaction mixture, shortly spin down the reaction mixtures and then incubate each reaction at 37°C for 1 hour to digest the
parental.
4.
Transformation and analysis
a.
Thaw one 100 μl DH5α competent cells for each transformation on ice.
b.
Transfer 5-10 μl Dpn I Digestion products directly into each DH5α competent cells and mix gently by pipetting.
c.
Incubate on ice for 30 minutes.
d.
Heat shock at 42°C for 90 seconds and immediately return transformation mix to ice for 2-3 minutes.
e.
Add LB medium (400-600 μl) to cells, mix gently, and incubate at 37℃for 1 hour while shaking at 220 rpm.
f.
Spread 100-200 μl cells onto agar plates containing the appropriate antibiotic for the plasmid vector. For the
mutagenesis and transformation controls, spread cells on LB-ampicillin agar plates containing X-gal and IPTG.
g.
Incubate overnight at 37°C.
h.
Select 3 to 5 colonies and analyze by plasmid isolation, PCR, or sequencing. DNA sequencing may be done to verify
that the desired mutations are successfully introduced and that no other mutations are found in the rest of the gene.
5.
Efficiency of mutagenesis
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a.
The mutagenic efficiency obtained using this method is consistently higher than 80%.
b.
The expected colony number from the transformation of the pUC19 White control mutagenesis reaction is ≥100
colonies. Greater than 80% of the colonies appear as blue colonies on agar plates containing IPTG and X-gal.
Troubleshooting Guide
Possible Cause
Comment & Suggestions
PCR product not visible
Insufficient template plasmid DNA
Increase the amount of template plasmid DNA used in mutagenesis PCR. Use
up to 50 ng plasmid DNA per 50 μl reaction.
Visualize the DNA template on an agarose gel to verify the quantity and
Poor template plasmid quality
quality. If the plasmid DNA is intact, there should be a brighter high molecular
weight band representing the supercoiled DNA topoform, very little of the open
circular and none of the linear conformation.
If the template plasmid DNA is GC-rich, please use the 5xGC-rich Buffer with
Difficult template plasmid DNA
MgSO4 instead of 10xPfu Buffer with MgSO4 in the reaction mixture. Make
sure that the final concentration is 1xGC-rich Buffer with MgSO4 in the reaction
mixture.
check the design, purity and concentration of the primers. Perform an
Primers not optimal
annealing temperature gradient to find the optimal annealing temperature of
the primers.
Only a couple or no bacterial colonies on the plate
The transformation efficiency of the
Check the transformation efficiency of the cells by transforming a supercoiled
competent cells is poor.
plasmid.
Transform low DpnI digestion products to the
Ethanol precipitate the Dpn I digested PCR product, and resuspend in a
competent cells.
decreased volume of water before transformation.
Spread low transformed cells on the agarose
Please enrich the transformed cells by low speed centrifugation and then
plates.
spread on the agarose plates
Large mutations lead to low numbers of
colonies
The concentration of antibiotics is too high
It is not uncommon to observe low numbers of colonies, especially when
generating large mutations. Most of the colonies that do appear, however, will
contain mutagenized plasmid.
Please use the proper concentration of antibiotics
Low mutagenesis efficiency with the sample reactions
Incompletely digest the parental plasmid DNA Allow sufficient time for the Dpn I to completely digest the parental template;
repeat the digestion if too much DNA template was present.
The primers may form the secondary
Increasing the annealing temperature up to 68℃ to alleviate secondary
structures inhibiting the mutagenesis reaction structure formation and improve mutagenesis efficiency.
Updated:07/2014
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