3 replies to this topic

[genetics3]

Hi! Two questions:
1. Do you know which is the content of blocking agent used before the hybridization step?
2. I know that patient and control DNA are mixed together, than cot dna, blocking agent and hybridization buffer are added to the mix. Then I have to
-Incubate at 95°C for 3 minutes.   
-Immediately Incubate at 37°C for 30 minutes.
Now the DNA is ready for the hybridization

My question is: during the incubation at 95°C the DNA is denaturated, but during the incubation at 37°C the DNA is renaturated?

Thank you so much!

[mdfenko]

agilent, at least, doesn't tell you what is in their blocking agent. the msds only identifies tris buffer (with exotic nomenclature). it claims there is nothing else hazardous in the block.

the dna will renature (the strands will anneal) at 37C.

[genetics3]

Thank you very much!Another clarification: if the dna renature at 37, how it can anneal to the single strand probes of the array?
I read in Agilent protocol that the array has to be incubated at 65 for 24 or 40 h (depending on the resolution of the array). At this point the dna return in single strand to hybridize to the single strand of the probes in the array?
Thank you so much!

[mdfenko]

the hybridization buffer contains components which facilitate hybridization during the 65C incubation.

Edited by mdfenko, 19 April 2012 - 11:04 AM.