Biomedical Engineering Reference
In-Depth Information
2.2 PCR condition
1st cycle: 94 °C 5 min
25-30 cycles: (94 °C 1 min +53 °C 1 min +72 °C 4 min) {
Note*2
}
Last cycle: 72 °C 7 min
3. Electrophoresis on an agarose gel (1-2 %) {
Note*3
}.
4. Apply 15-20
μ
L PCR product in each well, and cut gel pieces into tubes
{
Note*4
}.
5. Purify DNA fragments from agarose gel using a commercially available kit,
such as GENECLEAN Kit [QBiogene, cat#1001-200] {
Note*5
}.
6. Adjust volume to 100
L with dH
2
O.
7. Phenol extraction and ethanol precipitation.
μ
7.1 Add 100
L of PCI (phenol:chloroform:isoamyl alcohol 25:24:1) solu-
tion and vortex well.
7.2 Centrifuge at max speed of a benchtop centrifuge machine for 3 min, and
pipette off aqueous solution without disturbing or removing the pellet.
7.3 Transfer supernatant solution into a new tube avoiding the phenol layer.
7.4 Add 5
μ
L of 5 M NaCl.
7.5 Add 250
μ
L of 100 % EtOH.
7.6 Incubate the tube at −80 °C at least for 15 min.
7.7 Centrifuge at max speed (at over 15,000 rpm) for 15 min, and get pellet.
7.8 Rinse with 200
μ
L of 70 % EtOH.
7.9 Centrifuge at max speed of a benchtop centrifuge machine for 3 min, and
pipette off aqueous solution without disturbing or removing the pellet.
μ
7.10
Add
L RNase-free water, such as Pure Water (Invitrogen,
cat#10977-015).
20
μ
8. Use 1
L for checking of DNA concentration on an agarose gel.
9. Take another 1
μ
μ
L for DNA spectrometer, and adjust DNA concentration to
L {
Note*6
}.
10. Store the tube in a −20 °C freezer {
Note*7
}.
0.25
μ
g/
μ
Note
*1: Do not include more than 1
μ
g plasmid DNA. Nonspecifi c bands may be
amplifi ed.
*2: PCR cycles should be <30 to prevent amplifying nonspecifi c bands.
*3: Use thick and clean gels and fresh TAE buffer in the electrophoresis
equipment.
*4: It is important to cut gel pieces including PCR bands on an UV illuminator
as quickly to avoid DNA nicks by UV light.
*5: An alternative method to gel purifi cation is the QIAquick PCR purifi cation
kit from Qiagen.
*6: If the concentration of the DNA solution is low, adjust to 0.25
μ
g/
μ
L by
evaporation or EtOH precipitation.
*7: It is possible to store the purifi ed DNA solution for more than 3-4 years at
−20 °C.
