Protocol to generate plasmids with BsaI sites.
Moore Mutagenesis (variation developed by S. Moore)
Aka ‘round the horn PCR
Aka Full-plasmid PCR
Aka regular PCR
Here is the graphical outline:
Basically, you PCR around the entire plasmid. This gives you control over (1) large
deletions (by placing your primers to bracket the region you want to delete), (2)
Relatively large insertions (by including non-homologous regions on the 5’ ends of your
primers [colored red and blue above].
1. Design primers with similar melting temperatures. Generally 18-22 bases that are
complementary to the target plasmid. Extra regions to be inserted can be added to
the 5’ end of the primer.
2. Phosphorylate the primers. Regular primer synthesis leaves hydroxyl groups on
the 5’ end. These need to be phosphorylated in order for the ligase to work
efficiently. To phosphorylate the primers, first dissolve them in water at a
concentration of 50 uM.
a. Set up a phosphorylation reaction with the following components
i. NOTE: If making many reactions, it is good to make a master mix
with all components except primer (buffer, water, and PNK
enzyme).
ii. 2.5 uL T4 DNA ligase buffer (note the PNK buffer is for use in
radiolabeling procedures and does not have ATP)
iii. 3 uL of primer (total amount 300 pmoles)
iv. 19 uL water
v. 0.5 uL PNK (enzyme available from NEB)
b. Incubate at 37C for 30 minutes. Longer is OK.
3. PCR
a. Use an enzyme that does not add extra stuff to the 3’ end (e.g. do not use
Taq). Pfu and Pfusion work well.
b. Allow enough extension time for the polymerase to synthesize the entire
plasmid (6 minutes with Pfusion and plasmids up to 6 kb works well).
c. Example protocol
i. 1 uL miniprep plasmid
ii. 3 uL each phosphorylated primer (0.6 uM final)
iii. 3 uL polymerase buffer
iv. 0.6 uL dNTPs (25 mM each)
v. 0.5 uL Pfusion polymerase
vi. 22 uL water
d. cycling
i. 94 C for 2 minutes
Then 22 cycles of:
ii. 95 C for 30 seconds
iii. 50 C for 30 seconds
iv. 72 C for 6 minutes
4. Dpn I digest
a. Add 1 uL of Dpn I to PCR reaction and digest for 1 hour. This enzyme
only cuts methylated DNA. DNA produced inside bacteria (e.g. from a
miniprep) will be methylated whereas DNA produced by PCR will not.
Dpn I recognizes 4 bp and so cuts on average every 256 bases. This will
digest the parental plasmid into short fragments.
5. Analyze your 5 uL of your PCR reaction on an agarose gel and freeze the
remaining 25 uL (this is important to prevent overdigestion from the DpnI)
6. If the PCR reaction has one predominant band at the appropriate size, then
column purify your PCR product. If you don’t have a clean PCR product, you
may need to try increasing or decreasing the annealing temperature in your PCR
(try 45 or 55 C), or gel purify the appropriate product.
7. Ligation
a. Set up a ligation reaction with the following:
i. 1 uL T4 DNA ligase buffer
ii. 4 uL purified PCR product
iii. 4 uL water
iv. 1 uL T4 DNA ligase
b. Ligate at room temperature for 30 minutes
8. Transformation
a. Transform you ligated plasmid into bacteria and plate on appropriate
antibiotics
9. Grow up for miniprep.
10. Check plasmids by restriction analyses and Sanger sequencing.