I am a little confused as I see two different concentrations required to start sample prep of genomic DNA for solexa.
One states to start with genomic dna that is 100ng-1ug in concentration to shear (found here, page 63). The other (here, page 7) says to start with 1ug-5ug of purified DNA to shear.
My question is what is the starting concentration to use upon shearing the DNA?
My other question is, when you go to the end-repair step, won't the starting concentration be diluted down? So if I start with 1ug of genomic DNA, when I perform the end repair step, it will be 300ng (30ul/100ul * 1ug = 0.3ug) DNA concentration after end repair. Or if I start with 100ng of genomic DNA, it will be 30ng DNA concentation after end-repair. I am taking the enzymes into account here of course with the dilution. Even if I dilute with the water only (45ul), it would be 667ng or 66 ng respectively. Does it not matter what starting concentration I use for shearing because it will be diluted downstream anyway?
One states to start with genomic dna that is 100ng-1ug in concentration to shear (found here, page 63). The other (here, page 7) says to start with 1ug-5ug of purified DNA to shear.
My question is what is the starting concentration to use upon shearing the DNA?
My other question is, when you go to the end-repair step, won't the starting concentration be diluted down? So if I start with 1ug of genomic DNA, when I perform the end repair step, it will be 300ng (30ul/100ul * 1ug = 0.3ug) DNA concentration after end repair. Or if I start with 100ng of genomic DNA, it will be 30ng DNA concentation after end-repair. I am taking the enzymes into account here of course with the dilution. Even if I dilute with the water only (45ul), it would be 667ng or 66 ng respectively. Does it not matter what starting concentration I use for shearing because it will be diluted downstream anyway?
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