According to the protocol, 4 cycles 15/90 should give 950 bp fragments. My first try, I obtained fragments 500-900 bp in length (which worked well for library prep).
My second try with the same number of cycles (and same DNA concentration and volume), I have fragments that are 1200-1600 bp in length. My 'smear' on the gel is quite wide and falls within the correct range, but the highest DNA concentration falls in this high range.
Does anybody have a suggestion on how many cycles on/off more to get between 200-800 bp fragments needed for library prep? I don't want to accidentally shred them too small!
Many thanks!
There is some variable you are not accounting for (otherwise you would have obtained the same result) so you have to do it by trial and error.
Hi Ariana,
it actually depends on different parameters like the power setting of your machine, the volume of your sample, the concentration of your sample, the nature of your sample (pure DNA or cells for ChIP for example), the buffer you use on so on.
For ChIP experiments, starting from 300µl PFA fixed cells in 1.5ml eppendorff tubes, I usually use 10 cycles of 30s/30s at "high" power setting to get 300 - 800 bp fragments. For pure gDNA I guess you should lower the power and the number of cycles but as Alejandro stated, you may need to give different tries.
best
Dear Ariana,
Could you please specify which Br model do you use, which kind of tubes and sample volume?
You are working with DNA, not chromatin?
Best regards,
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