Hosuk:LabNotes/2014-3-24: Difference between revisions
Jump to navigation
Jump to search
>Hosuki78 (Created page with "*LabNote ===Home made CircLigase II Reaction Buffer=== *CircLigase II buffer is always run out fast. *Epicentre tech. suggested making buffer by myself. ...") |
>Hosuki78 No edit summary |
||
(5 intermediate revisions by the same user not shown) | |||
Line 8: | Line 8: | ||
====Reaction Buffer==== | ====Reaction Buffer==== | ||
*'''0.33M Tris-acetate + 0.66M Potassium acetate + 5mM DTT''' | *'''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]] |
Latest revision as of 19:19, 25 March 2014
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 |