Genomic DNA fragment labeling by random priming - (Mar/14/2008 )
I am looking for some advice on labeling of single stranded genomic fragments. Currently we use an Klenow-Fragment (exo) with random primers. We sonicate the DNA in order to decrease the fragments size. yielding 200-500bp fragments. It is my hope i can create random fragments Cy3/Cy5 labeled which are under 100bp. Could i use DNAse? but then how would i retrieve the small fragments. QIAttack PCR cleanup has trouble holding fragments under 200bp. I want to avoid phenol-chloroform. End labeling with terminal transferase has been considered. In all the goal of this is to create amplified random Cy labeld single strand fragments with high probe specificity and low background hybridization.
-Uwisc83-
QUOTE (Uwisc83 @ Mar 14 2008, 07:24 AM)
I am looking for some advice on labeling of single stranded genomic fragments. Currently we use an Klenow-Fragment (exo) with random primers. We sonicate the DNA in order to decrease the fragments size. yielding 200-500bp fragments. It is my hope i can create random fragments Cy3/Cy5 labeled which are under 100bp. Could i use DNAse? but then how would i retrieve the small fragments. QIAttack PCR cleanup has trouble holding fragments under 200bp. I want to avoid phenol-chloroform. End labeling with terminal transferase has been considered. In all the goal of this is to create amplified random Cy labeld single strand fragments with high probe specificity and low background hybridization.
I am not sure why exactly you want to do this, but have you considered treating chroamtin first with MNase (micrococcal nuclease)? It will cut the chromatin in inter-nucleosomal regions, and with optimal cutting conditions, you should achieve uniform ~146 bp size genomic fragments and some multiples thereof. Then you go ahead and do phenol-chloroform, followed by additional chloroform, etoh precipitation, followed by two rounds of 70% etoh wash. Your DNA will be golden for any use if you do it right. You can use glycogen during precipitation step, if I am not mistaken!
hth/
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