PCR of GC-rich sequence (E-cadherin)
Posted 11 July 2013 - 12:27 AM
- Protocol from normal one to slowdown PCR
- Mg concentration from 1,5 to 3 mM
- Polymerase without hot start or without
- Use of PCRx Enhancer solution from life tech (using this buffer it was possible to start amplifying samples without methylation)
- Amplify long (321bp) and short (172bp) sequences.
- Primer quantity from 0.2mM to 1.2mM
Right now I have no more ideas and I would like to ask you for some advice. Does someone work with this promoter and can give me some new directions?
Thanks you very much in advance
Posted 11 July 2013 - 01:11 AM
were you primers designed to amplify both methylated and un-methylated seq?
Posted 11 July 2013 - 06:47 AM
Posted 11 July 2013 - 07:15 AM
So, you are doing bisulfite sequencing PCR, right? How do your PCR cycling conditions look like? Can you post your primer sequences? Is the unmethylated DNA from Qiagen also bisulfite modified?
Posted 11 July 2013 - 08:06 AM
So, yes, I am doing bisulfite sequencing PCR.
1. I have tried different PCR protocols. Firstly a normal PCR about 50 cycles with 30 sec of denaturing, annealing and extension. Then I have tried to develop two different PCR protocols from these articles: Russell P. Darst et al 2010 and Ulrich H Frey et al 2008. The first one increase the times of denaturing,annealing and extension during the cycles and the second one defines a slowdown PCR.
2. The primers I mostly uses (the ones that have amplified the unmethylated sequence): f GTA ATT TTA GGT TAG AGG GTT AT, r CTCCAAAAACCCATAACTAAC
3. The control samples (methylated and unmethylated) are both bisulfite treated.
Posted 11 July 2013 - 08:30 AM
Posted 15 July 2013 - 07:29 AM