Half reactions for NexteraXT

[docbio]

Howdy all,

Wondering if anyone has had any success running "half reactions" of NexteraXT libraries or otherwise stretching those reagents? At the end of the library cleanup, if memory serves you only carry a little more than half of the reaction into bead based balancing. The bulk of our sequencing cost is in the actual library preparation steps, so cutting that in half is a tempting experiment but it would involve pipetting some very small volumes (0.5 ul). Does anyone have any experience with trying this?

Best,
docbio

[dfhdfh]

I've also thought about this. However, I think it's fine as is, since, as you already mentioned, you would need to pipet very small volumes. Additionally, I like to use the left-over of the library clean-up to do some QC (although you need only a few µls for that).

[kwaraska]

The Fluidigm C1 which is single cell cDNA uses 1/4 reactions so I believe it is tied to DNA/cDNA quantity. The only change in the 1/4 reaction is the first PCR step is 10 min instead of 5 min. I assume that is to give the tagmentation better working conditions since the volumes are so small.

Then we just use Tape Station to normalize the samples.

[docbio]

The Fluidigm C1 which is single cell cDNA uses 1/4 reactions so I believe it is tied to DNA/cDNA quantity. The only change in the 1/4 reaction is the first PCR step is 10 min instead of 5 min. I assume that is to give the tagmentation better working conditions since the volumes are so small.

Then we just use Tape Station to normalize the samples.

Interesting. This is useful info, I'm assuming the Fluidigm C1 protocol is online? I'm sure the concentration of DNA is critical to the tagmentation reaction. If we could move to half reactions it would save a lot of money. The only trick would be consistency. When we're normalizing for HiSeq (not going through bead based) the concentration of the finished libraries can vary somewhat.

Tape Station looks interesting. We're using Qubit or Quant-IT at the moment.

Best,
docbio

[docbio]

FYI, just as a follow up, we found that half reactions and even one quarter reactions work very well with NexteraXT in our hands, although the one quarter reaction size requires very precise pipetting that is only really amenable to individual pipetting instead of using a multichannel. We've stuck with half reactions and have great success in cutting costs while churning out good sequence data.

docbio

[Simone78]

if you are interested in making your own Tn5, you could take a look at our most recent paper: http://www.ncbi.nlm.nih.gov/pubmed/25079858
The plasmid is available from Addgene (ID 60240). Making the other reagents is really a piece of cake. The only thing we still buy from Illumina are the index oligos...and we dilute them 1:5! For inputs <1 ng DNA that amount is more than enough!