Hosuk:LabNotes/2014-3-24
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Home made CircLigase II Reaction Buffer[edit]
- CircLigase II buffer is always run out fast.
- Epicentre tech. suggested making buffer by myself.
Reaction Buffer[edit]
- 0.33M Tris-acetate + 0.66M Potassium acetate + 5mM DTT
Recipe of each material[edit]
1M Potassium acetate | pH 7.5 |
Potassium acetate | 9.82 g |
diH2O | x mL |
Total Volume | 100 mL |
1M Tris-acetate | pH 7.5 |
Tris-base | 12.1 g |
Acetic acid, glacial | 2.85 mL |
diH2O | x mL |
Total Volume | 100 mL |
CircLigase II Test Mix[edit]
- Add mix in test tube, incubate at 60C for 1hr., heat to deactivate ligase at 80C for 10min.
Test Mix | Final conc. | Volume |
ssDNA template | 10 pmole | 5 uL |
Reaction buffer, 10x | 1x | 2 uL |
MnCl2, 50 mM | 2.5 mM | 1 uL |
Betain 5 M | 1 M | 4 uL |
CircLigase II (5U/uL) | 5 U/uL | 1 uL |
Total Volume | 20 uL |
Result[edit]
- 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 (%)
Lane 1 | 2 | 3 | 4 | 5 | 6 |
Ladder | ssDNA | Original buffer | new MnCl2 | new Buffer | new MnCl2 & Buffer |
Conv. (%) | 92.2 | 91.8 | 92.2 | 92.6 |