Originally posted by atcghelix
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I routinely shear a couple hundred ng of DNA with a Covaris then concentrate it down to 20ul with an Amicon Ultra-0.5 10k. From there I can scale down my Kapa Hyper Prep reagents to 0.4x of what they should be. Haven't seen any problems yet! -
If you are going to do lots of library preps, it might be worth to do a comparison yourself. Prepping two libraries from the same input DNA and using 1/2 reactions with one and full reaction with other and comparison of PCR yield without sequencing would give some indications on performance of 1/2 reaction. With TruSeq kit, 1/2 and 1/3 reactions works as good as full reactions. Generally most kits use excessive amount of reagents to ensure good performance even with poor laboratory practices and low quality input.Leave a comment:
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Stretching Kapa library preps
Hi Everyone,
Has anyone tried doing half reactions with the Kapa library prep kits using 1000ng of input DNA? I'm curious if the amount of reagents in a half-reaction is enough to ligate adapters to 1000ng of input DNA.
Thanks very much!
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Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.07-01-2026, 11:43 AM
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I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.
Here are nine questions we think about, in roughly the order they matter, before...
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