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===V. Dialysis (dialyzer)=== '''This time did dialysis with 2 changes of 2X tn5 dialysis buffer, measured A280 to get molarity and then another 2 changes of tn5 storage buffer to concentrated and prepare for storage at -20C. Less dialysis steps can be used next time''' *1. Make 2X dialysis buffer and chill at 4C. Pool fractions with highest concentration. Test with biorad protein assay (bradford asssy) using 25 uL and 1 uL sample. '''pooled fractions 1-6, 7-10''' *2. Dialyze at 4C versus one change of 1 L 2x Tn5 dialysis buffer. Dialysis is for 2 hours with intermittent stirring every ~30 mins. (will concentrate sample some) *3. Measure A280 with nanodrop to get concentration (can take a small amount our of dialysis bag and leave the rest in, then don't have to change dialysis bags) *4. Dialyze at 4C with 2 changes of 1 Liter tn5 storage buffer (50% glycerol). Dialysis is for 2 hours with intermittent stirring every ~30 mins. (will concentrate sample ~2X) Tn5 dialysis buffer: 100mM Hepes, pH 7.2 0.2M NaCl 0.2mM EDTA 2mM DTT 0.2% TX100 20% Glycerol Tn5 storage buffer: 50 mM Hepes, pH 7.2 100 mM NaCl 0.1 mM EDTA 1 mM DTT 0.1% Triton X-100 50% Glycerol *5. Should be concentrated now, and in storage buffer (50% glycerol). Amicon filters results in protein denaturation and thus avoid using. Store at -20C. '''If not dialyzing with storage buffer''' *1. After dialysis if protein is concentrated enough an be used directly for transposome complex assembly. If not then put into storage buffer by adding 1.05 volume 99.5% glycerol and 0.33 volume of 2x Tn5 dialysis buffer (final 55% glycerol) and store at -20C. *2. (optional) If not concentrated enough, use Amicon Ultracel 30 filters and spin at 3000 rcf for 25 mins with mixing every 10 mins '''at 4C''' until final OD280 >3.0. Then can perform the above step of transposome complex assembly and preparation for storage at -20C. '''concentrating with filters greatly reduces specific activity of Tn5'''
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