Hi all,
Recently we started working on DNA hybridization. The DNA probe was adsorbed electrostatically onto cationic polymers. We have used 50mM tris-buffer with 150 mM of NaCl, but it looks there is no hybridization between the adsorbed probe and target. The same system is working well with colorimetric assay, so I think that tris-buffer is not suitable for DNA hybridization.
Regards
Hi Chaker,
The appropriate buffer, largely used for DNA and RNA hybridization, is the SSC buffer. You can either prepare it or just purchase the 20X concentrated one from sigma.
here the link for the buffer
http://www.sigmaaldrich.com/catalog/product/sigma/s6639?lang=en®ion=TN
All the best
Hi Makram,
Thanks for the answer Makram. I tried that buffer with different concentration 2x and 4x but it looks there is no hybridization. I think the probe and the target have a secondary structure which inhibits the hybridization. I will send you DNA probe and target sequences and you can help me to find the best temperature for hybridization.
Regards
you can find a good link for Tm calculations online
DNA Melting Temperature (Tm) Calculator -- EndMemo
Thanks Mohammed. I will check the EndMemo for Tm calculation. How are things with you in KSA?
The ssc should be diluted to 1X,. I will check the sequence. You may resolve the problem of the potential secondary structure by adding formaldehyde or Dmso. We will discuss in more details by email
All the best
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