as Touchdown, was also used to reduce the formation of spurious byproducts.
8 This involved setting the annealing temperature (T
a) 10 C higher than
the calculated primer melting temperature for the first cycle, then decreasing
it by 0.5 C every cycle for 20 cycles, and finally annealing at the T
m
during 9 subsequent cycles. When the reaction was carried out with primer
pair 341F (with GC clamp) and 534R, the annealing temperature (Touchdown)
was set at 60 C for the first cycle and decreased by 0.5 C each cycle
thereafter for 20 cycles, with the final 9 cycles performed at T
m of 50
C as shown below.
When the reaction was carried out with primer set 1055F and 1406R (with
GC-clamp), the annealing temperature was initially set at 65 C for the
first cycle and decreased by 0.5 C each cycle thereafter for 20 cycles,
with the final 9 cycles performed at Tm of 55 C as shown below.
In the final step of the reaction, the mixture was held at 75 C for
10 min to allow extension of incomplete products.9 Amplified products
were analyzed by agarose gel electrophoresis as follows: 5 ml product
was mixed with 1 ml gel loading buffer (40% sucrose, Mallinckrodt Inc.,
St. Louis, MO; 0.5% Brom Phenol Blue, Allied Signal Specialty Chemical,
Michigan Center, MI) and the mixture applied to a 1% agarose low melt
preparative grade gel (Bio-Rad Laboratories, Hercules, CA) in Tris-boric
acid-EDTA buffer (89 mM Sigma 7-9 Tris[hydroxymethyl]aminomethane, Sigma
Chemical Co, St. Louis, MO; 89 mM boric acid, Mallinckrodt Inc., St. Louis,
MO; 2 mM ethylenediamine tetraacetic acid, Fisher Scientific, Pittsburgh,
PA). Ethidium bromide (Bio-Rad Laboratori
'"/>
Source:
Page: All 1 2 3 4 5 6 7 8 9 10 11 12 13 Related biology technology :1.
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