Hosuk:LabNotes/2015-1-25: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Hosuki78
No edit summary
>Hosuki78
No edit summary
Line 72: Line 72:
***CircLigase II : 2 uL
***CircLigase II : 2 uL
***H2O : 63 uL
***H2O : 63 uL
 
**-->Run : 2 hr.





Revision as of 22:06, 25 January 2015

CA12k_Nov2014 : New Padlock Probes

Concentration

  • CA12k_Nov2014 that Matt has produced
    • V4 primers - CA12k_Nov2014_Contig
    • V7 primers - CA12k_Nov2014_Const


  • Produced at 2015-01-09
    • V4 : 10.7 ng/ul --> 10.7 ng/ul / (150nt*325Da/nt + 79Da) = 219nM (38ul)
    • V7 : 6.2 ng/ul --> 6.2 ng/ul / (150nt*325Da/nt + 79Da) = 127nM (18ul)
    • I’ve used 18uL of V4, and 18uL of V7 for the sample at 1/22
      • --> V4 : 103.7nM, V7 : 127nM in 100uL Ampligase Mix
      • --> 18.67 nM of V4, 22.86 nM of V7 in 100uL mix
      • Let’s fix the final concentration of Padlock probes to 100nM in 100uL Ampligase Mix


  • Produced at 2015-01-12
    • V7 : 28 ng/ul --> 28 ng/ul / (150nt*325Da/nt + 79Da) = 573nM (18ul)


  • Produced at 2015-01-15
    • V4 : 65.3 ng/ul --> 65.3 ng/ul / (150nt*325Da/nt + 79Da) = 1337nM (38ul)
    • V7 : 39.2 ng/ul --> 39.2 ng/ul / (150nt*325Da/nt + 79Da) = 803nM (18ul)


CA12k_Nov2014 - Test aminoallyl dUTP

  • I want to test anaspec’s a-dUTP that I’ve just got, and comparing to the result without a-dUTP


  • Sample : PGP1F, P13, cultured since 1/13/15


  • (1) 1/24 : Fix sample
    • Take out the dishes from CERC to the lab, and fix them with 10% Formaldehyde for 15min at 37C.
    • Wash with PBS 3 times, then incubate with 70% EtOH for 5 min, then 0.1N HCL for 1 min.


  • (2) 1/24 : Do RT ~ 15hr.
    • S1, S2 : with a-dUTP, 4mM, anaspec
    • S3, S4 : with a-dUTP, 2mM, life tech(?)
    • S5, S6 : no a-dUTP


  • (3) 1/25 : BS(PEG)9 (opened at 1/11/15), and Tris pH 8.0
    • S1, S2, S3, S4 only


  • (4) RNaseH : all samples
    • S1, S3, S5 : Run Ampligase with 100nM (final conc.) of CA12k_Nov2014_V4
    • S2, S4, S6 : Run 1st Rolony generation


  • (5-1) Ampligase Mix
    • Padlock probe : 2015-01-15 V4 : 1337nM (38ul)
    • -->100nM in final conc. --> 7.5uL in 100uL Ampligse Mix
      • Buffer : 10 uL
      • Ampligase (5U/uL) : 10 uL --> 0.5U/uL in final conc.
      • Padlock Probe (V4, 1/15/15) : 7.5 uL
      • H2O : 72.5 uL
    • -->Ampligase : 24 hr.


  • (5-2) Circligase II for 1st Rolony generation
      • Buffer : 10 uL
      • MnCl2 : 5 uL
      • Betaine : 20 uL
      • CircLigase II : 2 uL
      • H2O : 63 uL
    • -->Run : 2 hr.


  • (6-1) Run RCA : FISSEQ_RCA – S2, S4, S6 (1st rolony generation)
    • S4, S6 : 2mM a-dUTP, S2 : 4mM a-dUTP


  • (6-2) Run RCA : FISSEQ_ppRCA – S1, S3, S5 (V4 PP)
    • S3, S5 : 2mM a-dUTP, S1 : 4mM a-dUTP


      • Buffer : 10 uL
      • dNTP : 2 uL
      • a-dUTP : 2 uL for 4mM, 4uL for 2mM
      • Phi29 : 2 uL
      • H2O : 84 uL for 4mM, 82 uL for 2mM