Matt:LabNotes/2014-10-6: Difference between revisions

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(Created page with "==Testing Agi26k0gap + suppressor Oligos & Agi26k20gap== ===Sample=== *Agi26k0gap Control: PGP1F Fix 5.19.14 S2 *Agi26k0gap + suppressors: PGP1F Fix 5.19.14 S3 *Agi26k20gap: ...")
 
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| 10X Ampligase Buffer||10
| 10X Ampligase Buffer||10
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| [[Matt:LabNotes/2013-6-4 | Agi26k20gap]]||77
| [[Matt:LabNotes/2013-6-4 | Agi26k20gap]]||70
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| 25mM dNTP||1
| 25mM dNTP||1
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| 10 U/ul KlenTaq||2
| 10 U/ul KlenTaq||2
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| H2O||0
| H2O||7
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| Total||100
| Total||100
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*Incubate at 37C for 30 min
*Incubate at 37C for 30 min
*Incubate at 60C for 24 hrs
*Incubate at 60C for 24 hrs
*Wash with 1X PBS twice
*Add 200ul 0.5uM FISSEQ_ppRCA in 2X SSC + 30% formamide (pre-heated to 75C) and incubate at 45C for 30min
*Wash with 2X SSC twice
*Add 200ul RCA Mix and incubate at 30C for 16hrs
**H2O : 173uL
**10x Buffer : 20uL
**dNTP : 2uL
**a-dUTP : 4uL
**Phi29 (1000 U/µl) : 1uL
*Wash with 1X PBS once
*Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) at RT for 1hr
*Wash with 1X PBS twice
*Add 200ul 1M Tris pH 8.0 at RT for 30min
*Wash with 1X PBS twice and store in PBS
===Decoding Results===
*[[Hosuk:LabNotes/2014-10-20]]

Latest revision as of 00:44, 25 March 2015

Testing Agi26k0gap + suppressor Oligos & Agi26k20gap[edit]

Sample[edit]

  • Agi26k0gap Control: PGP1F Fix 5.19.14 S2
  • Agi26k0gap + suppressors: PGP1F Fix 5.19.14 S3
  • Agi26k20gap: Fix 10.1.14

Procedure[edit]

  • Agi26k0gap Control
Components Volume
5 U/ul Ampligase 10
10X Ampligase Buffer 10
Agi26k0gap 35
H2O 45
Total 100
  • Agi26k0gap + suppressors
Components Volume
5 U/ul Ampligase 10
10X Ampligase Buffer 10
Agi26k0gap 35
600 nM (100nM each) supp_Mix 5
H2O 40
Total 100
  • Agi26k20gap
Components Volume
5 U/ul Ampligase 10
10X Ampligase Buffer 10
Agi26k20gap 70
25mM dNTP 1
10 U/ul KlenTaq 2
H2O 7
Total 100
  • Incubate at 37C for 30 min
  • Incubate at 60C for 24 hrs
  • Wash with 1X PBS twice
  • Add 200ul 0.5uM FISSEQ_ppRCA in 2X SSC + 30% formamide (pre-heated to 75C) and incubate at 45C for 30min
  • Wash with 2X SSC twice
  • Add 200ul RCA Mix and incubate at 30C for 16hrs
    • H2O : 173uL
    • 10x Buffer : 20uL
    • dNTP : 2uL
    • a-dUTP : 4uL
    • Phi29 (1000 U/µl) : 1uL
  • Wash with 1X PBS once
  • Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) at RT for 1hr
  • Wash with 1X PBS twice
  • Add 200ul 1M Tris pH 8.0 at RT for 30min
  • Wash with 1X PBS twice and store in PBS

Decoding Results[edit]