CHIP-on-chip - (Jan/02/2007 )
Does anybody here know why affy CHIP-on-chip protocol is doing fragmentation before the labeling step?
Thanks!
I think it has something to do with the labelling efficiency in combination with the length of sequences of the array. Each spot is a 35mer tiled across unique sequence so it would make sense to have your IP DNA fragmented to a smaller size to increase hybridisation efficency. Also smaller fragments are labelled more effectively.
Thanks!
I tried affy protocol for amplification and didn't work well (incorporated dUTP to the DNA so it can be fragmented by UDG later on). WGA kit works really well for the DNA amplification. Is there another way to fragment DNA to less than 100bp if I am not using dUTP?
hn37041,
how did you determine the affy protocol did not work very well? by gel electrophoresis? you may not see the small fragments by this method.
N
It is the amplification yield is too low. I can only get a couple of ug after amplification, affy requires 9ug.
I found that DNaseI might work for the fragmentation if I am not using dUTP.
I tried affy protocol for amplification and didn't work well (incorporated dUTP to the DNA so it can be fragmented by UDG later on). WGA kit works really well for the DNA amplification. Is there another way to fragment DNA to less than 100bp if I am not using dUTP?
Did you use Affy's protocol as recommended or you modified? It seems that the primer concentration of 100uM (4 uM final) too much to me.
I used affy protocol. Didn't really try to modified their protocol. Love the WGA kit!