Tn5 transposase inactivated by high pressure?

[Simone78]

Hi!
I was wondering is someone has some experience with dispensing a mix containing Tn5 transposase (such as the one in the Nextera kit, for example) with a liquid handling robot at "high" pressure (2 bar) or higher than atmospheric pressure.
While I always get good libraries in a tube reaction (even down to 1-2 ul for the tagmentation, compared to 20 ul recommended by Illumina) I NEVER got a library when using our liquid dispenser and performing the reaction in the same volume.
Is it the pressure? Is the inactivation due to the fact that the Tn5 is going through the (metal) tips of the robot twice, first from the reservoir to the tips and then from the tips into the plate?
It seems that the problem is not due to adsorption of the Tn5 to the plastic. Coating the wells with 0.5% BSA didn´t help, as well as addition of a bunch of additives to improve the efficiency of the reaction (PEG, Ficoll, dextran, DMF, etc etc).
Any help is very much appreciated!

[ericchow]

Can you decrease the aspiration and dispense rates to lower the pressure?

Are these tips new each time or are they resused from run to run since they're metal? Can you pipet some of the Tn mix through your system and then use it in the tube format to see if it's an issue of going through the metal tips?

[Simone78]

Can you decrease the aspiration and dispense rates to lower the pressure?

Are these tips new each time or are they resused from run to run since they're metal? Can you pipet some of the Tn mix through your system and then use it in the tube format to see if it's an issue of going through the metal tips?

I´m already decreasing the aspiration and dispense speed to 1/5 of what I do when I use other reagents/enzymes. In another test I decreased the pressure to 8 psi but it didn´t get better at all. I got the same broad peak and very little DNA after 20 cycles of enrichment PCR!

I´m using metal tips, washing them both on the inside and outside between different dispensing rounds. Interestingly, KAPA HiFi DNA Pol, Superscript II (and IV) or DNA Polymerase are not affected when dispensed at high pressure.

Thanks a lot for your suggestion about using the Tn5 dispensed with the robot in a tube reaction. I´ll certainly do it soon!

[kobeho24]

The TTP Labtech's Mosquito is kind of nanoliter liquid handler, some researchers have already performed Nextera XT on their hand with this dispensing platform in a final volume of 4ul for each. And if I am not making mistakes, the tip is plastic but not metal. Of course, I am not sure about the aspiration/dispensing pressure of this machine. But I think there might be issues with the tips. Maybe there is a iron sensitive domain in Tn5? I'm not sure. Perhaps you can ask the technician of TTP.http://ttplabtech.com/posters/mosqui...equencing-ngs/

[Simone78]

A tagmentation done on the robot showed to be 50-100 times LESS efficient that the same one done in a 0.2 ml tube. But flushing the tips with water for 1 min made it already much better. It really seems that residues of detergent on the inside of the tips are inactivating the Tn5! I´ll try now to use a milder detergent and wash the tips much longer and see what happens.

The TTP Labtech's Mosquito is kind of nanoliter liquid handler, some researchers have already performed Nextera XT on their hand with this dispensing platform in a final volume of 4ul for each. And if I am not making mistakes, the tip is plastic but not metal. Of course, I am not sure about the aspiration/dispensing pressure of this machine. But I think there might be issues with the tips. Maybe there is a iron sensitive domain in Tn5? I'm not sure. Perhaps you can ask the technician of TTP.http://ttplabtech.com/posters/mosqui...equencing-ngs/

probably it works on the Mosquito because you use disposable tips and don´t have residues of detergent. This Mosquito is a really interesting machine, thanks for sharing!

[lab.lady]

Has anyone yet tried to perform Nextera XT on any of the mosquito liquid handlers? I would like to know your thoughts if so!