If you did not line up two pieces of felt together, then pin the template to the second piece of felt and cut it out as well.
This approach not only verifies proper treatment of DNA but also provides an adequate template for the second round of PCR, in which methylated cytosines are differentiated from unmethylated ones by applying methylated CpG dinucleotides specific primers.
The fragment of desired size was gel purified and used as a PCR template for the second round of PCR amplification.
One-fiftieth of each first-round PCR product was then used as a template for the second round of PCR with the following primers: F2 (5'-GCTCTGAGGAATGGGTTACAGGAG-3') and intR2 (5'-CCCAATCATGGCCGACAAAA-3').
A PCR product containing the lpoA Δ58-252) allele was then generated via overlap extension ('stitching') PCR (Heckman and Pease, 2007), using PCR primers ANG235 x ANG239 and ANG241 x ANG242 with E. coli genomic DNA as template for the first round of PCR and ANG235 x ANG242 with first-round-PCR-products as template for the second round of PCR.
The normalized cDNA was used as a template for the second run of LD-PCR.
Therefore, the sequence encoding Tx-Xyn-AF7 was used as the template for the second round of epPCR.
The product was diluted 50 times and then used as template for the second round of PCR, which involved NUP and outer/inner gene-specific primers.
The cRNA was purified (RNeasy, Qiagen) and used as template for the second round of double-stranded cDNA synthesis with random primers (Invitrogen).
In the first step, amplification of a cre fragment was carried out with 5′-AGCACCTGGGCCAGCTCAACAT-3′ and 5′-TGCGCAGCAGGGTGTTGTAG-3′ to generate a template for the second round of amplification with cre-specific primers used for animal genotyping.
After thermocycling, the PCR1 reactions were diluted with 20 μl H2O and 1 μl was used as template for the second round of PCR, which was carried out in the same 2 × 384 wells format as the first PCR, however, with different sets of primers which are internal to the first set (nested).
