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Old 03-16-2015, 12:19 PM   #1
Degraz
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Default TrueSeq PCR free POOR LIGATION efficiencies

Is anyone else seeing poor ligation efficiencies for Truseq PCR free preps? Our BA and quBit values are high and qPCR values are very low.
<10% ligation efficiency.

Can anyone help?
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Old 03-19-2015, 10:05 AM   #2
lac302
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Are you seeing the correct size on the BA trace?

I had pretty low yield on the last pcr free library I made, a little over 1nM. Loaded ~6pM and still got around 10Gb (V3, 2x275).
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Old 03-19-2015, 12:16 PM   #3
Chipper
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What is the normal efficiency?
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Old 04-08-2015, 05:03 AM   #4
FWOS
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Default

I've seen the same thing. Some folks are doubting the adapters and adding double (5ul of 15uM index per reaction). From what i have tested it looks like ligation efficiency is much better with low input (100-200ng). higher input seems to drastically impact the results even when swapping out ligation mixes from other manufacturers, which performed better but did not result in satisfactory ligation efficiencies.

Has anyone had issues with the atailing mix? wondering if the dATPs are limiting or the Klenow is crap.

Best,
FWOS
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