Hosuk:LabNotes/2014-3-24: Difference between revisions

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====CircLigase II Test Mix====
====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
{| {{table}} border = 1
| align="center" style="background:#f0f0f0;"|'''Test Mix'''  
| align="center" style="background:#f0f0f0;"|'''Test Mix'''  
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====Result====
====Result====
*Based on the intensity measurement, and comparing to original buffer sample, new buffer is 85% efficiency, not bad.
*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
*new 50mM MnCl2 is made from 1M MnCl2


 
*Conversion % = (Circligased)/(CircLigase+ssDNA template) x 100 (%)
{| {{table}} border = 1
{| {{table}} border = 1
| align="center" style="background:#f0f0f0;"|'''Lane 1'''  
| align="center" style="background:#f0f0f0;"|'''Lane 1'''  
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| 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" | Ladder || align="center" | ssDNA || align="center" | Original buffer || align="center" | new MnCl2 || align="center" | new Buffer || align="center" | new MnCl2 & Buffer
|-
|-
| align="center" | normalized || align="center" |  || align="center" | '''100''' || align="center" | '''89.7''' || align="center" | '''79.3''' || align="center" | '''84.9'''
| align="center" | Conv. (%) || align="center" |  || align="center" | '''92.2''' || align="center" | '''91.8''' || align="center" | '''92.2''' || align="center" | '''92.6'''
|}
|}


[[File:CircLigaseIIBufferTest_edited_2014-03-24.png|450px]]
[[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

File:CircLigaseIIBufferTest edited2 2014-03-24.png