FIRST STEP: MICRODISSECTING
Materials:
Metaphase (Store in fixative in-20°C),
Fixative (Methanol : glacial acetic acid is 3:1, fresh prepared)
1.2-mm capillaries (World Precision Instruments, Sarasata FL, 1B1 20F4)
24×60mm coverslips,
2% Trypsin (Lifetechnologies)
Giemsa stain (Lifetechnologies, Grandisland, NY, 10092-013)
Topoisomerase I (Promega Corp., Madison, WI, M2851)
5×Topo buffer (200mmol/L Tris-HCl (pH7.5), 100mmol/L MgCl2, 250mmol/L NaCl)
10×dNTP (0.2mM) (100mM dNTP(Pharmacia LKB, Piscataway, NJ, 27-2045-01) diluted by water)
DOP-PCR primer (UN1: 5’-CCGACTCGAGNNNNNNATGTGG-3’),diluted to 100uM
Procedure: 1.Warm metaphase to room temperature;
2.Centrifuge at 1000rpm for 5min, discard the supernatant;
3.Wash the palette twice with fresh made fixative,
4.Drop the metaphase on the coverslip, airdry.
5.Incubate at 37°C for 3 days.
6.Trypsinize the metaphase and then stain at 37.5% Giemsa stain for 5 minutes.
7.Air dry. Incubate at 37°C overnight.
8.Glass needles are prepared on a automatic pipette puller(Narishige, Japan, PC-10)
9.Mix the following to prepare collection buffer
|
Reagents |
Volume (ul) |
|
Topo buffer |
2 |
|
0.2mM dNTP |
2 |
|
UN1 primer |
0.4 |
|
dH2O |
16 |
|
Topoisomerase I |
0.4 |
|
Total volume |
~20 |
Aliquots the collection buffer to 4 PCR tubes, 5ul each.
10.Place the coverslip on a inverted microscope, the glass needle is controlled by a hydraulic micromanipulator (Narishige, Japan, MO-302)
11.Under the microscope, dissect the region which you are interested in, and casually transfer it into the collection buffer, place on ice.
12.Dissect 4~8 copies of interested DNA, then incubate the PCR tubes on PCR machine at 37°C for 1 hour.
