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

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>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. ...")
 
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====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====
{| {{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 is 85% efficiency, not bad.
*new 50mM MnCl2 is made from 1M MnCl2
{| {{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" | 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|600px]]

Revision as of 03:36, 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