Hosuk:LabNotes/2013-6-10: Difference between revisions

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>Hosuki78
(Created page with "=====Training Day 1===== *Some of important things *Phi29 enzyme from '''Enzymatics''' is used. *Actually Jay likes to use (or trusts) Enzymatics, so almost all reagents are f...")
 
>Hosuki78
 
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=====Training Day 1=====
*[[Hosuk:Lab_Notes|LabNote]]
 
 
====Training Day 1====
*Some of important things
*Some of important things
*Phi29 enzyme from '''Enzymatics''' is used.
*Phi29 enzyme from '''Enzymatics''' is used.
*Actually Jay likes to use (or trusts) Enzymatics, so almost all reagents are from Enzymatics.
*Actually Jay likes to use (or trusts) Enzymatics, so almost all reagents are from Enzymatics.
*The concentration of Phi29 of Enzymatics that Jonathan has used is '''100U/uL''' which is '''High concentration one''' from Enzymatics, and this concentration is the same as low concentration one from Epicentre.
**'''1uL of phi29 in 200uL total mix'''
**I could try 3uL of phi29
*RNase H step after RT is critical step, so do this carefully.
*RNase H step after RT is critical step, so do this carefully.
*RNase H + Riboshredder mix is
**'''10uL of RNase H''' +
**'''2uL of Riboshredder''' +
**'''20uL of 10x Buffer''' +
**168uL of H2O
*Try HeLa cells unless I get rolonies with PGP1F.
*Try HeLa cells unless I get rolonies with PGP1F.
*The sample (that I've got some signal at 05/29) doesn't seem to have actual rolonies,
*There are no dots, just too much even signal distribution.
*Jonathan fixed cells just using 10% Formamide
**Aspirate media
**Add 50uL of 10% Formamide (200uL for glass bottom)
**Incubate for 15min
**Wash with PBS three times
**Store in Ethanol
=====Rolony Construction=====
*S1 : +RT, +CircLigase, +RCA
*S2 : -RT (no RT enzyme), +CircLigase, +RCA
*Jonathan's samples in 96 well (2 positive, 2 negative controls)
*Jonathan fixed his cells in 96 well plate, using 10% Formalin and incubating 15min, without 37% Formalin.
*HCL incubation for 1min.
*Run RT for overnight (5:00pm)

Latest revision as of 18:20, 14 June 2013


Training Day 1[edit]

  • Some of important things
  • Phi29 enzyme from Enzymatics is used.
  • Actually Jay likes to use (or trusts) Enzymatics, so almost all reagents are from Enzymatics.
  • The concentration of Phi29 of Enzymatics that Jonathan has used is 100U/uL which is High concentration one from Enzymatics, and this concentration is the same as low concentration one from Epicentre.
    • 1uL of phi29 in 200uL total mix
    • I could try 3uL of phi29


  • RNase H step after RT is critical step, so do this carefully.
  • RNase H + Riboshredder mix is
    • 10uL of RNase H +
    • 2uL of Riboshredder +
    • 20uL of 10x Buffer +
    • 168uL of H2O


  • Try HeLa cells unless I get rolonies with PGP1F.


  • The sample (that I've got some signal at 05/29) doesn't seem to have actual rolonies,
  • There are no dots, just too much even signal distribution.


  • Jonathan fixed cells just using 10% Formamide
    • Aspirate media
    • Add 50uL of 10% Formamide (200uL for glass bottom)
    • Incubate for 15min
    • Wash with PBS three times
    • Store in Ethanol



Rolony Construction[edit]
  • S1 : +RT, +CircLigase, +RCA
  • S2 : -RT (no RT enzyme), +CircLigase, +RCA
  • Jonathan's samples in 96 well (2 positive, 2 negative controls)
  • Jonathan fixed his cells in 96 well plate, using 10% Formalin and incubating 15min, without 37% Formalin.
  • HCL incubation for 1min.


  • Run RT for overnight (5:00pm)