146
YOSHIO KANEMITSU AND TAKEHIKO KOJI
1 cycle:
40-50 cycles:
1 cycle:
94 c e
94°e
55°e
nee
noe
4°e
1 mins
40 secs
40 secs
40 secs
3 mins
continuous
3. Check the PCR products by electrophoresis.
4. Correct the solutions in one tube, add same volume of buffered phenol and vortex briefly. Centrifuge 5 mins at 5000 rpm
and save upper aqueous phase.
5. - 10. is same as the first PCR.
Alternate protocol
It is possible to produce ssDNA by single step asymmetric PCR
using a pair of primers, one of which is added in vast excess over
the other. The concentration of one primer is from 5 to 10 nM,
while that of the other is 500 nM. Other conditions ofPCR are the
same as the first PCR of two-step asymmetric PCR.
T-T dimeriza- Dilute the ss cDNA solution to 50 f-lg/ml and put it in a flat sition of DNA liconized quartz dish with a light path of 0.5 mm. Irradiate it by a
UV lamp (254 nm) at 5.0 J/m 2 sec for 2000 secs. (See Chapter 11)
DNA frag- This step may be abbreviated if the permeability of the probe is
mentation by sufficient, for example, in case the probe length is shorter than
S1 endo- 250 bases. Otherwise, digest the probe with SI nuclease in order
nuclease to reduce the size.
1. Mix the following:
50 ~g/ml ss cDNA probe
lOx Sl nuclease buffer
200 nUll Sl nuclease
YOSHIO KANEMITSU AND TAKEHIKO KOJI
1 cycle:
40-50 cycles:
1 cycle:
94 c e
94°e
55°e
nee
noe
4°e
1 mins
40 secs
40 secs
40 secs
3 mins
continuous
3. Check the PCR products by electrophoresis.
4. Correct the solutions in one tube, add same volume of buffered phenol and vortex briefly. Centrifuge 5 mins at 5000 rpm
and save upper aqueous phase.
5. - 10. is same as the first PCR.
Alternate protocol
It is possible to produce ssDNA by single step asymmetric PCR
using a pair of primers, one of which is added in vast excess over
the other. The concentration of one primer is from 5 to 10 nM,
while that of the other is 500 nM. Other conditions ofPCR are the
same as the first PCR of two-step asymmetric PCR.
T-T dimeriza- Dilute the ss cDNA solution to 50 f-lg/ml and put it in a flat sition of DNA liconized quartz dish with a light path of 0.5 mm. Irradiate it by a
UV lamp (254 nm) at 5.0 J/m 2 sec for 2000 secs. (See Chapter 11)
DNA frag- This step may be abbreviated if the permeability of the probe is
mentation by sufficient, for example, in case the probe length is shorter than
S1 endo- 250 bases. Otherwise, digest the probe with SI nuclease in order
nuclease to reduce the size.
1. Mix the following:
50 ~g/ml ss cDNA probe
lOx Sl nuclease buffer
200 nUll Sl nuclease