Bisulfite/sequencing-based Methylation Analysi - (Apr/07/2014 )

I decreased the extension temp to 65 for 90'' and last extension for 20 minutes. Still no Products, no smeer! I also increased the cycles number to 40.

Have you tried lowering your annealing temperature? This is the first thing to try if you are getting no amplification, and I asked previously if you had tried it.

Yes I did. I used to lower Ann Temp. 50 and 55.
The scary thing is that I didn't see smeer down the gel?

I am sure I am adding the templates, primers and dNTPs!!
Why no smeer even?!

This is the DNA fragment I am interrogating:

CATAAGAGACGCCCTCAAAACACTATGACTTTTATTAGTTATTCACCTCCCCAGAGCTGTCCCTGGATACAGACAACATAGGTATGAGGTAGGGGGTACGTGGAGCCAAACAGGAGGTAATACCTTCTGAATTTAGATGCTAACAAGAAAACATGGGGAAAGGTGGCCCAGATACACTAGGCCCTTTATTCTTTGGGCCTGTAACACCTACTTATTTGATTGTGGCATGAACCATGAACTCGGTTTGGGGCAAGTCCTTCCTTTTTCTGCAGTCTGTGGAATCGGGAGAGGTTAGCCATTGCCGCCTCTATTCACCTTAGGCATGATGTAAACAGAAATTAGTATCTCTGCCTCCTTCCTTTTTCCACACCCCGAAGTCATTTCCTCTTAACCTGGGATTTCGACGTCTATATTCCCTCTGTATGATAGATGCACTCAGGGAGGCAAGGGGGGGAGGGAGGAACTTCTTAAAATTCCCCCAGAATGTTTTGACACTAGTTTTCAGTGTTGCAATTGAGACTAGTCAGTTTCTACTTTGGGTTTCCATCAGAAAGTTCTGTAGGAGTAGAGTATATAAGCACCAGGAGCAGCCAAGGCAGCAGAAGGAACAGTGGGTGTCCAGGCACATCAGACCAGGCAGCTCGCAGCAAAGCAAGGTAAGTTCTCTCCTCTTCCCTGTCGCTAACTCCCTGCATCTAGAGGCTGTCCAGATTCAGACTCCAGGGGACAGGCTACCCCTGAACCAGGCAGCGTGGGAGTGGGGTAAGTGGATTCTGGGAG

Left primer: 

5'-TGGAGTTAAATAGGAGGTAATATTTTTTG-3'

 

Right primer:

5'-CCCCTTACCTCCCTAAATACATCT-3'