usual unsuccessful pcr

[polymerase1]

hi guys,

am trying to amplify a constructed cassette within the pIGCN21 plasmid with 2 a bit long primers. indeed, they r 21 mer each but there is an extra 42 mer attached as they'll be a homologus recombination at later stage.

the primers are previously designed, i've just attached the 42 nt to each of them. i'm using the iProof master mix which contains all the required recipes including (1.5 mM of MgCl2).

no products were obtained within all the following protocols:
98C 30s
98C 10s
55C 30s
72C 60s
72C 10 min
4C hold

i've changed the annealing temp. several times (from 55, 53, 60, 72) nothing obtained.

also, i did two different cycling conditions (15 cycles with 70C and 20 cycles with 45C) once again nothing was obtained.

tried to change the MgCl2 concentration (from 1.5 mM to 1.8, 2.1, 2.4, 2.7 and 3 mM) and i did the last protocol (two different cycling conditions) again nothing obtained. no bands at all except primer dimers.

i'm preparing a touchdown protocol to go with but anyone have got any other idea that i may go through???????

thanks.

[danfive]

Yeah try more cycles. How about a control of the 21mer primers without the new 42nt addition? Sort of a control to see amplification.

Also how about premix big primers and template DNA (cassette) perform melt and anneal step on thermocycler, chill on ice. Quickly add rest of PCR mix and begin amplification with extension step, followed by regular PCR cycles.