Thermo cycling consisted of an initial denaturation at 94AdegC for 2 min followed by 35 cycles of denaturing at 94AdegC for 1 min, primer annealing (58-65AdegC) for 2 min and primer extension
(72AdegC) for 2 min.
2 ul water free nuclease for full length 16S rRNA gene amplification and initially was denatured at 94degC for 2 min followed by 30 cycles consisting of denaturation at 94degC for 60 s, primer annealing at 55degC for 60s and primer extension
at 72degC for 3 min and a final extension at 72degC for 10 min using multigene thermal cycler.
The conditions used in one step RT-PCR were include reverse transcription with the Incubation at 45degC for 45 min, initial denaturation at 95degC for 5 min, and 40 cycle each of 95degC for 40 sec, annealing at 56degC for 1 min, primer extension
at 72degC for 30 sec followed by final extension at 72degC for 10 min.
The SNaPshot primer extension
products are separated with
The PCR amplification conditions were initial denaturation at 94[degrees]C for 3 min followed by 40 cycles of denaturation at 94[degrees]C for 1 min, primer annealing at 35[degrees]C for 1 min, primer extension
at 72[degrees]C for 2 min, and a final primer extension
at 72[degrees]C for 5 min.
First, primer extension
is performed using site-specific primers and a combination of dNTPs and ddNTPs, selected so that different alleles will result in products of different and predicted sizes.
During this work, we found that this measuring system was not able to detect changes in methylation levels during patient treatment, and so 2 different primer extension
schemes were investigated.
observed that the single mismatches present at the last five positions from the primer 3 end could totally block the primer extension
PCR tubes with 50 uL reaction mixture were placed in the PCR thermocycler (Gene Amp Biosysytem-9700, Applied Bio-system) and following temperature cycling conditions were programmed; initial denaturation at 94@C for 5 min, followed by 40 cycle denaturation at 94oC for 1 min, annealing at (35oC) for 1 min, primer extension
at 72@C for 1 min and final extension temperature 72@C for 5 min.
was performed using a SNaPshot ddNTP Primer Extension
Kit (Applied Biosystems, Foster City, CA).
It also does not require Target Specific Primer Extension
(TSPE), signal amplification and does not feature a washing step.
The PCR cycle involves three steps: denaturation, primer annealing, and primer extension