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]
- Using Mattek dish from Hosuk:LabNotes/2014-3-3 that was fixed in 4% formalin
- Strip off dye-probes Hosuk used with 80% formamide pre-heated to 75C and incubated @45C for 15min
- Wash with 1X PBS twice
- 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]
- 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[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
- dcProbe0 and dcProbe1 are from Illumina, dcProbe2 is from IDT
- 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
- Goal was successful