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

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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" | normalized || align="center" |  || align="center" | '''100''' || align="center" | '''89.7''' || align="center" | '''79.3''' || align="center" | '''84.9'''
|}
|}


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

Revision as of 03:37, 25 March 2014

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

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

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

  • Based on the intensity measurement, and comparing to original buffer sample, new buffer is 85% efficiency, not bad.
  • new 50mM MnCl2 is made from 1M MnCl2


Lane 1 2 3 4 5 6
Ladder ssDNA Original buffer new MnCl2 new Buffer new MnCl2 & Buffer
normalized 100 89.7 79.3 84.9

File:CircLigaseIIBufferTest edited 2014-03-24.png