Mastercycle gradient

Sections of paraffin-embedded tissues deposited in a tube were subjected to the
deparaffinization process and enzymatic digestion with proteinase K (200 mg/ml) at 56
°C for 2-4 days. After digestion, DNA was extracted using the phenol-chloroform
method recommended by Sambrook²² .
After extraction and purification, the DNA samples were subjected to PCR, using the
primers PCO3 and G74 (oligonucleotide primers used in PCR amplification reactions),
which amplify 100 base pairs (bp) of the human b-globin gene^{1 (link)}, with the aim of evaluating the
adequacy and integrity of the DNA present in each sample.
The positive samples were subjected to PCR with generic primers for HPV
(GP5+/GP6+^{8 (link)}), capable of
amplifying 140 bp of the HPV L1 gene.
To demonstrate that there was no contamination by exogenous DNA, a negative control
was used, containing all the reagents of the mixture except for the DNA. A lineage of
HeLa cells containing DNA from integrated HPV 18^{26 (link)} was used as the positive control.
The amplifications were performed in an Eppendorf thermocycler, Mastercycle gradient
(Germany). Forty cycles of amplification were effected applying 1 min for
denaturation at 95° C, 1 min for annealing at 55° C and 1.5 min for the chain
elongation at 72° C.
The amplification products, also known as amplicons, were analyzed on 7%
polyacrylamide gel stained with silver.