Library Construction Format

[Isequencestuff]

Hey Guys,

I'm rather new to library construction & sequencing and am unsure of a few things. A little background: I'm using the Truseq sample preparation kit for library workflow, and my colleague is using the KAPA Biosystems Library preparation kit. We're doing low throughput construction (about 5 libraries per construction) and I'm wondering what format is generally best to use as the tube and magnets go. I've been reading that some people use 1.5 mL microcentrifuge tubes and others use PCR tubes both with their corresponding magnets. PCR tubes fit into our thermocycler for controlled incubations while 1.5 mL tubes do not, does everyone follow the exact incubation temperatures? Whats the best approach to deciphering what format that I should use? Or what is the most common tube/magnet used?

[kwaraska]

Yes-the incubation temps are very important as the enzymes work best at that temp. I use strip tubes and that magnet and my libraries look great.

I suggest using whichever format gives you the best control over temperature, which sounds like the 0.2ml PCR tubes

[Isequencestuff]

Alright, 0.2 mL PCR strips sound ideal since incubation temperatures are vital. Can you recommend a magnet that has worked best for you? If you're using AMPure beads to purify, can you fit the entire end repair reaction along with 160 ul beads into a PCR tube?

[kwaraska]

magnet

We got our mag plates from Agencourt Biosciences some years ago. I think they were bought by Beckman Coulter so I would try there.

The adding 160 of beads step is very close and you have to be careful when mixing but surface tension is a wonderful thing.

[mnkyboy]

I have had good success using v-bottom plates with the AMPure and the Ambion magnet. That magnet is compatible with PCR plates too if you prefer those.

I prefer the Sarstedt v-well bottom plates.

[Isequencestuff]

Thanks for the magnet and tube recommendations. I'll look into both of those magnets as well as my surgical pipetting skills when it comes to End Repair purification. PCR strips seem good for low throughput library construction, any worry over contamination? It seems easy for DNA to jump to another adjacent well with removal of their caps.

[kwaraska]

I just am very careful when removing the caps. Obviously spin down the tubes before you open them.

I don't cap them with the beads in there-that should reduce the possibility of cross contamination there.

[Isequencestuff]

I just am very careful when removing the caps. Obviously spin down the tubes before you open them.

I don't cap them with the beads in there-that should reduce the possibility of cross contamination there.

Ah I see, thanks for the recommendations. I'll give them a try.