Exo-CIP
PCR
cleanup
protocol
This protocol is
used to
clean PCR products for direct sequencing. It utilizes the action of two
enzymes
to remove free dNTP’s and excess primers remaining from PCR.
Exonuclease I
(Exo) is an enzyme that will cut up single-stranded DNA such as primers
and
unfinished PCR products. Calf Intestinal Phosphatase removes dNTPs that
were
not incorporated into the PCR DNA. This protocol is not suitable to
prepare
samples for A-overhang (TA/pGEM) cloning.
Procedure
1. For each PCR
reaction,
add 0.2 µl of
Exonuclease (ExoI) to a 1.5 ml tube.
2. Add an equal
amount of
Calf Intestinal Phosphatase (CIP) to the same tube.
3. Add 0.4 µl of the Exo-CIP mixture to each PCR
reaction tube
and mix by pipetting.
4. Incubate 15
min at 37° C followed by 15 min inactivation
incubation at 85° C.
5. Dilute the
PCR product
1:1 with sterilized DI water. The sample is now ready for
direct
sequencing.
Materials
necessary
Fresh
PCR product in 0.2 ml PCR tube
Exonuclease
1
Calf
Intestinal
Phosphatase
1.5 ml tube
10 µl pipette tips
Sterilized
DI water
Equipment
required
2 µl
micropipette
Thermocycler
or heat block