Matt:LabNotes/2014-3-4: Difference between revisions

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#Strip off dye-probes Hosuk used with 80% formamide pre-heated to 75C and incubated @45C for 15min
#Strip off dye-probes Hosuk used with 80% formamide pre-heated to 75C and incubated @45C for 15min
#Wash with 1X PBS twice
#Wash with 1X PBS twice
#Anneal Padlock Probes @45C for 24hrs
#Anneal Padlock Probes @45C for 24hrs (Put in at noon, some bubbles and hard to keep on middle glass)
#*1X Ampligase Buffer
#*1X Ampligase Buffer
#*2uM of each padlock probe
#*2uM of each padlock probe
Line 32: Line 32:
| Total||200
| Total||200
|}
|}
====Secondary RCA====
#Aspirate but don't wash
#'''Pre-annealing the FISSEQ_ppRCA primer''' for the rolling circle amplification reaction.
##1ul of RCA primer(200 uM, GATATCGGGAAGCTGA*A*G) in 199ul of '''2xSSC/30% formamide'''
##Preheat primer at 60C (Tm = 55C when '''not''' in 30% formamide)
##Add 200ul and incubate at 45C for 15min.
##Aspirate, and wash using 2x SSC twice
##Aspirate, and wash using 0.1x SSC twice
#Prepare the rolling circle amplification reaction mixtures on ice.
##171 ul ddH2O,
##20  ul 10x Phi29 buffer,
##2  ul 25 mM dNTP,
##4  ul 2 mM aminoallyl dUTP
##3  ul Phi29 DNA polymerase (Epicentre, low concentration)
##*200ul Total Volume
#Add and incubate 20hrs at 30C. (Put in at 1pm 3/5/14)
#Wash 1X PBS once
#Add 200ul BS(PEG)9 mix to each well and incubate @RT for 1hr
#Wash 1X PBS twice
#Add 200ul 1M Tris pH 8.0 @RT for 1hr
#Wash 1X PBS twice
====Detection====
*Make 100ul of 1uM dcProbe0-Cy3, dcProbe1-Cy3, and dcProbe2-Cy3 in 30% formamide and 2X SSC
**dcProbe0 and dcProbe1 are from Illumina, dcProbe2 is from IDT
***Need to order more dcProbe1-Cy3
*Checked dcProbe0
*Strip
*Checked dcProbe1
*Strip
*Checked dcProbe2
**Dust fell into dish? Lots of non-specific binding so can't see secondary rolonies anymore
===Results===
*Using PISA7 Paramters: Gaussian std (3), upper bound (-2e-4), area upper (50), area lower (4), axratio lower (.6), circ upper (1.6), circ lower (.8), perim conn (8), bkgmult lower (7)
'''dcProbe0'''
*ACTB 2nd Rolonies
**Pos1: 108
**Pos2:
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Gain 680'''
| align="center" style="background:#f0f0f0;"|'''Gain 620'''
|-
| 130 2nd Rolonies||244 2nd Rolonies
|-
| [[File:Pos2_noEGF_dcProbe0_680_Cy3_ch00_MIP_PISA7Overlay.png|450px]]||[[File:Pos2_noEGF_dcProbe0_620_Cy3_ch00_MIP_PISA7Overlay.png|450px]]
|}
'''dcProbe1'''
*RAB7A 2nd Rolonies
**Pos2:
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Gain 680'''
| align="center" style="background:#f0f0f0;"|'''Gain 620'''
|-
| 595 2nd Rolonies||779 2nd Rolonies
|-
| [[File:Pos2_noEGF_dcProbe1_680_Cy3_ch00_MIP_PISA7Overlay.png|450px]]||[[File:Pos2_noEGF_dcProbe1_620_Cy3_ch00_MIP_PISA7Overlay.png|450px]]
|}
'''dcProbe2'''
*Didn't analyze because of dust
===Conclusion===
*The goal was to see if we could get ACTB and RAB7A 2nd Rolonies
**Goal was successful
***There is no problem with dye-probes from Illumina or ppACTB/ppRAB7A
***[[Matt:LabNotes/2014-2-25#Experimental_Sample_.28E6_.26_F6.29|Previously]] the lack of ACTB and RAB7A 2nd Rolonies must have been due to low amount of mRNA in fixed sample

Latest revision as of 17:47, 10 March 2014

3 Genes Secondary Rolonies Mattek Dish[edit]

  • Hosuk got much more primary rolonies (incl. ACTB and RAB7A) by culturing PGP1F without EGF (Hosuk:LabNotes/2014-3-3)
  • Since Matt:LabNotes/2014-2-25#Detection resulted in many MALAT1 secondary rolonies but few ACTB or RAB7A
    • This is possibly due to sample having very few ACTB or RAB7A
    • Also possible the dyes we got from Illumina (dcProbe0-Cy3 and dcProbe1-Cy3) have somehow degraded since MALAT1 detection was using a new dye-probe from IDT

Procedure[edit]

  1. Strip off dye-probes Hosuk used with 80% formamide pre-heated to 75C and incubated @45C for 15min
  2. Wash with 1X PBS twice
  3. Anneal Padlock Probes @45C for 24hrs (Put in at noon, some bubbles and hard to keep on middle glass)
    • 1X Ampligase Buffer
    • 2uM of each padlock probe
    • 0.5 U/ul Ampligase
Component Volume
10X Ampligase Buffer 20
200uM ppMALAT1 2
200uM ppACTB 2
200uM ppRAB7A 2
5U/ul Ampligase 20
H2O 154
Total 200

Secondary RCA[edit]

  1. Aspirate but don't wash
  2. Pre-annealing the FISSEQ_ppRCA primer for the rolling circle amplification reaction.
    1. 1ul of RCA primer(200 uM, GATATCGGGAAGCTGA*A*G) in 199ul of 2xSSC/30% formamide
    2. Preheat primer at 60C (Tm = 55C when not in 30% formamide)
    3. Add 200ul and incubate at 45C for 15min.
    4. Aspirate, and wash using 2x SSC twice
    5. Aspirate, and wash using 0.1x SSC twice
  3. Prepare the rolling circle amplification reaction mixtures on ice.
    1. 171 ul ddH2O,
    2. 20 ul 10x Phi29 buffer,
    3. 2 ul 25 mM dNTP,
    4. 4 ul 2 mM aminoallyl dUTP
    5. 3 ul Phi29 DNA polymerase (Epicentre, low concentration)
      • 200ul Total Volume
  4. Add and incubate 20hrs at 30C. (Put in at 1pm 3/5/14)
  5. Wash 1X PBS once
  6. Add 200ul BS(PEG)9 mix to each well and incubate @RT for 1hr
  7. Wash 1X PBS twice
  8. Add 200ul 1M Tris pH 8.0 @RT for 1hr
  9. Wash 1X PBS twice

Detection[edit]

  • Make 100ul of 1uM dcProbe0-Cy3, dcProbe1-Cy3, and dcProbe2-Cy3 in 30% formamide and 2X SSC
    • dcProbe0 and dcProbe1 are from Illumina, dcProbe2 is from IDT
      • Need to order more dcProbe1-Cy3
  • Checked dcProbe0
  • Strip
  • Checked dcProbe1
  • Strip
  • Checked dcProbe2
    • Dust fell into dish? Lots of non-specific binding so can't see secondary rolonies anymore

Results[edit]

  • Using PISA7 Paramters: Gaussian std (3), upper bound (-2e-4), area upper (50), area lower (4), axratio lower (.6), circ upper (1.6), circ lower (.8), perim conn (8), bkgmult lower (7)

dcProbe0

  • ACTB 2nd Rolonies
    • Pos1: 108
    • Pos2:
Gain 680 Gain 620
130 2nd Rolonies 244 2nd Rolonies
File:Pos2 noEGF dcProbe0 680 Cy3 ch00 MIP PISA7Overlay.png File:Pos2 noEGF dcProbe0 620 Cy3 ch00 MIP PISA7Overlay.png

dcProbe1

  • RAB7A 2nd Rolonies
    • Pos2:
Gain 680 Gain 620
595 2nd Rolonies 779 2nd Rolonies
File:Pos2 noEGF dcProbe1 680 Cy3 ch00 MIP PISA7Overlay.png File:Pos2 noEGF dcProbe1 620 Cy3 ch00 MIP PISA7Overlay.png

dcProbe2

  • Didn't analyze because of dust

Conclusion[edit]

  • The goal was to see if we could get ACTB and RAB7A 2nd Rolonies
    • Goal was successful
      • There is no problem with dye-probes from Illumina or ppACTB/ppRAB7A
      • Previously the lack of ACTB and RAB7A 2nd Rolonies must have been due to low amount of mRNA in fixed sample