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*[[Hosuk:Lab_Notes|LabNote]] ===Home made CircLigase II Reaction Buffer=== *CircLigase II buffer is always run out fast. *Epicentre tech. suggested making buffer by myself. ====Reaction Buffer==== *'''0.33M Tris-acetate + 0.66M Potassium acetate + 5mM DTT''' ====Recipe of each material==== {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''1M Potassium acetate''' | align="center" style="background:#f0f0f0;"|'''pH 7.5''' |- | align="center" | Potassium acetate || align="center" | '''9.82 g''' |- | align="center" | diH2O ||align="center" | '''x mL''' |- | align="center" | Total Volume ||align="center" | '''100 mL''' |} {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''1M Tris-acetate''' | align="center" style="background:#f0f0f0;"|'''pH 7.5''' |- | align="center" | Tris-base || align="center" | '''12.1 g''' |- | align="center" | Acetic acid, glacial ||align="center" | '''2.85 mL''' |- | align="center" | diH2O ||align="center" | '''x mL''' |- | align="center" | Total Volume ||align="center" | '''100 mL''' |} ====CircLigase II Test Mix==== *Add mix in test tube, incubate at 60C for 1hr., heat to deactivate ligase at 80C for 10min. {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Test Mix''' | align="center" style="background:#f0f0f0;"|'''Final conc.''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | align="center" | ssDNA template || align="center" | '''10 pmole''' || align="center" | '''5 uL''' |- | align="center" | Reaction buffer, 10x ||align="center" | '''1x''' || align="center" | '''2 uL''' |- | align="center" | MnCl2, 50 mM ||align="center" | '''2.5 mM''' || align="center" | '''1 uL''' |- | align="center" | Betain 5 M ||align="center" | '''1 M''' || align="center" | '''4 uL''' |- | align="center" | CircLigase II (5U/uL) ||align="center" | '''5 U/uL''' || align="center" | '''1 uL''' |- | align="center" | ||align="center" | Total Volume || align="center" | '''20 uL''' |} ====Result==== *Based on the intensity measurement, and comparing to original buffer sample, new buffer showed similar efficiency to the original buffer. *new 50mM MnCl2 is made from 1M MnCl2 *Conversion % = (Circligased)/(CircLigase+ssDNA template) x 100 (%) {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Lane 1''' | align="center" style="background:#f0f0f0;"|'''2''' | align="center" style="background:#f0f0f0;"|'''3''' | align="center" style="background:#f0f0f0;"|'''4''' | align="center" style="background:#f0f0f0;"|'''5''' | align="center" style="background:#f0f0f0;"|'''6''' |- | align="center" | Ladder || align="center" | ssDNA || align="center" | Original buffer || align="center" | new MnCl2 || align="center" | new Buffer || align="center" | new MnCl2 & Buffer |- | align="center" | Conv. (%) || align="center" | || align="center" | '''92.2''' || align="center" | '''91.8''' || align="center" | '''92.2''' || align="center" | '''92.6''' |} [[File:CircLigaseIIBufferTest_edited2_2014-03-24.png|450px]]
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