Applied Biosystems GeneAmp PCR System 9700, User Manual

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Supplied Methods B-5
Touchdown PCR
When the optimal annealing temperature is unknown, one strategy, touchdown PCR,
incrementally decreases the annealing temperature in early cycles in order to
maximize the yield of specific products.
This supplied method has an initial annealing temperature (65 °C) that incrementally
decreases by an additional 0.5 °C in each of the first 20 cycles, followed by 10 cycles
at 55 °C.
XL PCR
XL PCR is the protocol specified for amplification of 5 kb-40 kb PCR products, using
rTth DNA Polymerase, XL, and unique reaction conditions.
This protocol uses two-temperature cycling (94 °C for 15 seconds; 68 °C for
10 minutes) and invokes a 15 second AutoX (automatic segment extension) for the
anneal/extend step in the last 12 cycles.
By providing longer templates, XL PCR complements technologies for rapid,
long-range PCR. More complete genes can be amplified in one reaction from known
expressed sequences, thus more introns can be crossed. You can use XL PCR for the
amplification of the control target, a 20.8 kb product from Lambda DNA, supplied in
the kit.
This process is further described in the product insert for the GeneAmp ® XL PCR Kit
(P/N N808-0192).
2 Tmp x 10 2 Tmp x 20
94.0
0:15
65.0
0:30
94.0
0:15
55.0
0:30
*
Start Return
Method: Touchdown PCR
F1 F2 F3 F4 F5
2 Holds 2 Tmp X 16 2 Tmp X 12 1 Hld
94.0
1:00
94.0
0:15
68.0
10:00
94.0
0:15
68.0
10.00
72.0
10.00
4.0
Start Return
Method: XL PCR
F1 F2 F3 F4 F5
*