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!
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!
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