[nucacidhunter]

[QUOTEThe only difference is the entry DNA, since they are amplicons, the concentration is a lot lower. On average around 200 ng, which is less than the recommended 1µg-5µg input. Should I alter the quantities of the reagents? ][/QUOTE]

I am not aware of any current kit requiring 1-5 ug input DNA, although few years ago it was common for shotgun library prep kits. In Illumina's TruSeq Nano DNA kit which requires 100-200 ng input depending on intended insert size, after size selection, end repair and A tailing around 20-50 ng DNA is usually remains for ligation step. With that input amount one can prepare good libraries with half reactions. You can use 20-30 ng of amplicons with half reactions from A tailing step onward. I would recommend cleaning up your amplicons before A tailing for good results.

[Neiltje]

Well, both the kits from NEB and KAPA start with 1-5 µg start DNA.
I'm planning on tracking my samples on the bioanalyzer after every step the first time I do this, so I can see what happens with my amplicons.

[nucacidhunter]

Well, both the kits from NEB and KAPA start with 1-5 µg start DNA.
I'm planning on tracking my samples on the Bioanalyzer after every step the first time I do this, so I can see what happens with my amplicons.

You are right about those kits and you approach is reasonable. With Nano kit after every clean up step I also run them on Bioanalyser and quantified the output with ds PicoGreen reagent (for more accuracy) and that is how I worked out the DNA going into ligation step.

[Neiltje]

Yes, use less adapters that's actually very important sorry I forgot to mention this!

I check my A-tailed libraries on the Qubit and use a 20:1 molar ratio of adapters. The idea is to maximise the efficiency of ligation and minimise formation of adapter dimers.

Most kits use adapter stocks at 15 micromolar. If you are not certain just reduce/dilute the adapter by the same ratio as your input DNA compared to recommended input DNA.

Just to make sure: you only adjust the adaptor concentration, never the concentration of the enzymes used in the different steps?