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Old 02-25-2016, 01:31 PM   #1
jlove
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Location: Boston, MA

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Default Assay to test for ligation efficiency?

Hi,
Has anyone got a method for assessing TruSeq ligation efficiency, pre-PCR? We use the KAPA Illumina library quant kit for final library quantification, and I can certainly try using this, but I am wondering if anyone knows about actually using the results to assess the efficiency? I'm not sure how to evaluate the results to determine next steps.
Thanks in advance!
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Old 02-26-2016, 09:48 AM   #2
jdk787
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Quote:
Originally Posted by jlove View Post
Hi,
Has anyone got a method for assessing TruSeq ligation efficiency, pre-PCR? We use the KAPA Illumina library quant kit for final library quantification, and I can certainly try using this, but I am wondering if anyone knows about actually using the results to assess the efficiency? I'm not sure how to evaluate the results to determine next steps.
Thanks in advance!
You could try ligating, then quantifying your sample with Qubit to determine the total amount of DNA present.
Then quantify the same sample with the KAPA quantification kit which will only measure the amount of ligated material in your sample.
Ligation efficiency can then be determined by comparing the Qubit number to the Kapa number.
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Old 02-26-2016, 11:03 AM   #3
HESmith
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Be aware that the KAPA assay will detect adapter dimers as well as bona fide library clones. While dimers are the product of ligation, I suspect that's not what you want to measure :-).

The TruSeq kit contains inline controls for the enzymatic steps, including ligation, so you may want to incorporate those into your protocol.
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Old 02-26-2016, 11:16 PM   #4
RNA_
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If your lab is licensed for radioactive work, prepare a test library in parallel using trace amounts of 32P-labeled substrate spiked into your sample --> run on PAGE with appropriate radiolabeled size markers --> phosphorimaging.
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