Hi everyone,
I have just made my first library for sequencing. As i didn't have much material, after the initial gel purification I did 15 rounds of PCR, followed by a second gel purification as I had a lot of adapters coming through. My final library has the correct size distribution with no additional bands however the concentration is very low. The bioanalyser (high sensitivity DNA chip) results put it at 48.77pg/ul or 242.5pM but the q-PCR (KAPA sybr fast) results give me 140pg/ul or 710pM.
Is the difference that I see between the two quantitation methods due to the low amount of starting material?
Also should I amplify the library some more by doing another 3 rounds of PCR? Or is there enough there to send to the sequencers?
Any help would be greatly appreciated!
Thanks,
Melanie
I have just made my first library for sequencing. As i didn't have much material, after the initial gel purification I did 15 rounds of PCR, followed by a second gel purification as I had a lot of adapters coming through. My final library has the correct size distribution with no additional bands however the concentration is very low. The bioanalyser (high sensitivity DNA chip) results put it at 48.77pg/ul or 242.5pM but the q-PCR (KAPA sybr fast) results give me 140pg/ul or 710pM.
Is the difference that I see between the two quantitation methods due to the low amount of starting material?
Also should I amplify the library some more by doing another 3 rounds of PCR? Or is there enough there to send to the sequencers?
Any help would be greatly appreciated!
Thanks,
Melanie
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