Site Directed Mutagenesis Protocol
1. Determine the amino acids you wish to alter.
2. Design a primer sequence that will introduce the desired codon(s) change as well
as introduce a new restriction site. The easiest way to do this is to using The
Primer Generator (http://www.med.jhu.edu/medcenter/primer/primer.cgi).
Instructions are available on the site.
3. Design the primer such that either side of the mutation(s) has a melting temp (Tm)
of 40-45˚C. Count C or G as 4˚C, T or A as 2˚C and add up the bases. Always end
the primers in C or G even if this means raising the melting temperature.
4. Design the second primer so that it is the reverse complement of the first primer
Example:
The gene sequence is: ATG CCC AAA GGG GGA GCC CCC TGT GGG TAG.
You wish to change the alanine (GCC) to a serine.
The Primer Generator site tells you that changing the GCC-> TCC will
accomplish this, and introduce a BamHI site (GGATCC).
Good primers would be:
5’-GCCCAAAGGGGGATCCCCCTGTGGG-3’
5’-CCCACAGGGGGATCCCCCTTTGGGC-3’
This gives 42 (4+4+4+4+2+2+2+4+4+4+4+4+2) and 40
(4+4+4+4+4+2+4+2+4+4+4) degrees of melting temperature on the sides of the
mutation.
5. Set up the PCR reaction by adding the components to a tube on ice in this order:
Water
36µl
DNA (miniprep)
5µl
Primer 1 (10µm)
1µl
Primer 2 (10µm)
1µl
dNTPs (20mM)
1µl
Pfu Buffer
5µl
 pipet to mix well after this step
Pfu (2.5U/µl)
1µl
 mix well again
6. PCR program (hot start):
95˚C 5 min.
95˚C 30 sec.
18x
55˚C 1 min.
68˚C 2 min./kb + 2 min. (eg: for an 8kb plasmid = 18 min..)
68˚C 2 min./kb + 2 min.
7. When the PCR reaction is complete, transfer 5µl to a fresh tube. Add 1µl Dpn1 to
the remaining 45µl, pipetting to mix, and incubate 1-2 hr. at 37˚C.
8. Run 5µl pre and 5µl post Dpn1 digestion product on an agarose DNA gel. If SDM
is successful, a similar size plasmid band will appear in both lanes, containing the
undigested and digested DNA.
9. After you have verified SDM by successful Dpn1 digest, transform 5µl of the
digest into 100µl chemically competent XL1 blue E. coli cells. Other competent
cells may be substituted. Plate on appropriate selective media (usually LB Amp).
Incubate overnight at 37˚C and expect 10-100 colonies.
10. Select 4 colonies and inoculate 5ml overnight liquid cultures. Grow at 37˚C.
11. Isolate miniprep DNA. Digest plasmids with the appropriate enzyme to diagnose
the presence of the desired mutations. Always digest the starting plasmid (i.e.,
SDM template for PCR in step 5) in parallel as a control.