Stretching Kapa library preps

[atcghelix]

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!

[nucacidhunter]

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.

[bilyl]

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!

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!