S7804 datasheet(20/32 Pages) ETC

20 / 32 page

The amount of reagents required in each reaction is:

10X Universal PCR buffer

2.5 µL

2.5 mM dNTP Mix**

2.0 µL

P or M primers (white, red)

2.0 µL

"Hot start" enzyme (5 U/µL)

0.1 µL (0.5 U)

dH2O

16.4 µL

23.0 µL

Template DNA (50 ng/µL)

2.0 µL

TOTAL VOLUME

25.0 µL

** Upon first use, make aliquots of 2.5 mM dNTPs, which should be freeze-

thawed no more than 5 times.

c. Aliquot 23 µL of each "master mix" (white and red) into corresponding PCR

tubes.

d. Add:

2 µL of water to the no DNA control tube.

2 µL of modified sample DNA to each of the sample tubes.

2 µL of modified normal DNA control to each control tube.

e. Place tubes in the thermocycler block, and perform PCR under the following

conditions:

Denature:

for "hot start" enzyme check manufacturer's specifications

Then, perform 35 cycles of the following conditions:

denature

95°C / 30 seconds

anneal

62°C / 30 seconds

extend

72°C / 30 seconds

Final Extension:

72°C /10 minutes

f. Remove the tubes from the thermocycler block. From this point on, it is

important to designate separate pipettors and work areas for amplified vs.

unamplified samples. This prevents carry-over contamination of future DNA

samples with the amplified product.

S7804 Datasheet(PDF) 20 Page - List of Unclassifed Manufacturers