SECOND STEP: PROBE AMPLIFICATION AND LABELING
Materials:
T7 Sequenase (US Biochemicals, Cleveland, OH, 70702)
Enzyme dilution buffer (obtain from the T7 sequenase kit, aliquots to 7ul per tube, store at -20°C)
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
AmpliTaq® DNA Polymerase, LD (Perkin-Elmer, Foster City, CA, N808-0157)
10×PCR reaction buffer (100mmol/L Tris-HCl (pH 8.4), 40mmol/L MgCl2, 500mM KCl, 0.01%Gelatin)
Biotin-16-dUTP or Spectrum Orange or other fluorescence dye (1mM).
3M sodium acetate (pH5.2)
cold absolute ethanol
Procedure:
1.Sequenase is diluted eight-fold by add 1ul sequenase to 7ul enzyme dilution buffer.
2.Synthesis of primer-terminated copies. This is achieved by 6~8 cycles of denaturation, primer annealing, and low temperature primer extension using a highly processive polymerase-Sequenase. The dissected products are heated to 94°C for 5min, then In each priming cycle, the temperature are 94°C for 1min, 30°C for 3min, and 37°C for 2min. A 0.2ul portion of diluted sequenase is added to the tube at each period of 30°C incubation.
3.The primer-terminated DNA strands are then amplified in a PCR reaction.
Mix the following according to the recipe here:
|
Reagents |
Volume (ul) |
|
10×PCR buffer |
5 |
|
0.2mM dNTP |
5 |
|
UN1 primer |
1 |
|
dH2O |
40 |
|
AmpliTaq® DNA Polymerase, LD |
0.5 |
|
Total volume |
~50 |
Add the mixture to the tube. The PCR cycle is: 94°C for 5min, then followed by 30 cycles of 94°C 1min, 56°C 1min, and 72°C 1.5min. The products are finally extended at 72°C for 5min. Thus the first-round of PCR is finished. Reagents Volume (ul) 10×PCR buffer 5 0.2mM dNTP 5 UN1 primer 1 Biotin-16-dUTP or Spectrum Orange or other fluorescence (1mM) 1 dH2O 40 AmpliTaq® DNA Polymerase, LD 0.5 Total volume ~50
4.The first-round PCR products are amplified in another PCR reaction which is done by adding 2ul first-round PCR product to the similar system of first PCR. The second PCR is 25 cycles. And the temperature condition is similar to those of first PCR.
5.Test PCR efficiency by agarose gel electrophoresis.
6.If the PCR is good, label the PCR products in another PCR reaction that contain fluorescence as follows:
8.Incubate for 2hr at -20°C.
9.Use FISH test your chromosome microdissection. (See Protocol for FISH)
