Matt:LabNotes/2014-5-27: Difference between revisions

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(Created page with "==Artifical Rolonies== ===Procedure=== #Add 140ul 100nM MALAT1_template to each well #*Pre-heat to 85C #*Incubate @RT for 30 min #**C4 & C5 Incubate @RT for '''15 min''' #Aspi...")
 
>Mzcai
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#Add 140ul 100nM MALAT1_template to each well
#Add 140ul 100nM MALAT1_template to each well
#*Pre-heat to 85C
#*Pre-heat to 85C
#*Incubate @RT for 30 min
#*Incubate @RT for '''75 min'''
#**C4 & C5 Incubate @RT for '''15 min'''
#**C4 & C5 Incubate @RT for '''60 min'''
#Aspirate & Wash once with 1X PBS
#Aspirate
#Capture with 10pM ppMALAT1  
#Capture with 100pM ppMALAT1
#*Incubate @45C for 24 hours (5:30p- )
#*Incubate @45C for 24 hours (6:30p- )
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Component'''
| align="center" style="background:#f0f0f0;"|'''Component'''
Line 14: Line 14:
| 10X Ampligase Buffer||80
| 10X Ampligase Buffer||80
|-
|-
| 1nM ppMALAT1||80
| 2nM ppMALAT1||40
|-
|-
| 5 U/ul Ampligase||80
| 5 U/ul Ampligase||80
|-
|-
| H2O||560
| H2O||600
|-
|-
| Total||800
| Total||800
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===Procedure===
===Procedure===
*Strip with 80% formamide for 15min @RT
*Use pre-made artificial MALAT1 rolonies from [[Matt:LabNotes/2014-5-16]]
*Wash with ddH2O twice
**Strip with 80% formamide for 15min @RT
**Wash with ddH2O twice


'''Experimental & Negative Control''' (Positions 3-12)
'''Experimental & Negative Control''' (Positions 3-12)

Revision as of 01:40, 28 May 2014

Artifical Rolonies

Procedure

  1. Add 140ul 100nM MALAT1_template to each well
    • Pre-heat to 85C
    • Incubate @RT for 75 min
      • C4 & C5 Incubate @RT for 60 min
  2. Aspirate
  3. Capture with 100pM ppMALAT1
    • Incubate @45C for 24 hours (6:30p- )
Component Volume
10X Ampligase Buffer 80
2nM ppMALAT1 40
5 U/ul Ampligase 80
H2O 600
Total 800

Exo I/III Test

Procedure

  • Use pre-made artificial MALAT1 rolonies from Matt:LabNotes/2014-5-16
    • Strip with 80% formamide for 15min @RT
    • Wash with ddH2O twice

Experimental & Negative Control (Positions 3-12)

Component Volume
10X Ampligase Buffer 50
2uM ppMALAT1 2.5
H2O 447.5
Total 500

Positive Control (Positions 1-2)

Component Volume
10X Ampligase Buffer 10
H2O 90
Total 100