Matt:LabNotes/2014-2-25: Difference between revisions
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===Procedure=== | ===Procedure=== | ||
====Anneal Padlock Probes==== | |||
*1X Ampligase Buffer | *1X Ampligase Buffer | ||
*2uM of each padlock probe | *2uM of each padlock probe | ||
Line 10: | Line 10: | ||
*Incubate @ 45C for 24 hours | *Incubate @ 45C for 24 hours | ||
**Wrapped in parafilm and put in ziploc with wet towel | |||
====Fixed Template - 25U Ampligase (C9 & F9)==== | =====Fixed Template - 25U Ampligase (C9 & F9)===== | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Component''' | | align="center" style="background:#f0f0f0;"|'''Component''' | ||
Line 27: | Line 28: | ||
|} | |} | ||
====Fixed Template - 50U Ampligase (C10 & F10)==== | =====Fixed Template - 50U Ampligase (C10 & F10)===== | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Component''' | | align="center" style="background:#f0f0f0;"|'''Component''' | ||
Line 43: | Line 44: | ||
|} | |} | ||
====1st Rolony (E6 & F6)==== | =====1st Rolony (E6 & F6)===== | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Component''' | | align="center" style="background:#f0f0f0;"|'''Component''' | ||
Line 63: | Line 64: | ||
|} | |} | ||
====Empty Negative Control (D7 & E7)==== | =====Empty Negative Control (D7 & E7)===== | ||
*Not going to waste Ampligase since it's highly unlikely anything will be ligated, but will do RCA in these wells | *Not going to waste Ampligase since it's highly unlikely anything will be ligated, but will do RCA in these wells | ||
====Pre-circularized Padlock Probes (C7 & C8)==== | =====Pre-circularized Padlock Probes (C7 & C8)===== | ||
*Combined all the pre-circle ppMALAT1 made on: [[Matt:LabNotes/2014-2-10]] into | *Combined all the pre-circle ppMALAT1 made on: [[Matt:LabNotes/2014-2-10]] into 64ul | ||
*75% EtOH for 10 minutes | *75% EtOH for 10 minutes | ||
*Wash with 1X PBS 2 times | *Wash with 1X PBS 2 times | ||
*0.1N HCl for 2 minutes | *0.1N HCl for 2 minutes | ||
*Wash with 1X PBS 3 times | *Wash with 1X PBS 3 times | ||
*Add | *Add 32ul to C7 & C8 | ||
====Fixed Pre-circularized Padlock Probes (F7 & F8)==== | =====Fixed Pre-circularized Padlock Probes (F7 & F8)===== | ||
*Not going to do anything during Ampligase step | *Not going to do anything during Ampligase step | ||
====Secondary RCA==== | |||
#Aspirate each well but don't wash | |||
#*F7 well was dried up | |||
#'''Pre-annealing the FISSEQ_ppRCA primer''' for the rolling circle amplification reaction. | |||
##3ul of RCA primer(200 uM, GATATCGGGAAGCTGA*A*G) in 597ul of '''2xSSC/30% formamide''' | |||
##Preheat primer at 60C (Tm = 55C when '''not''' in 30% formamide) | |||
##Add 50ul to each well, 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. | |||
##513 ul ddH2O, | |||
##60 ul 10x Phi29 buffer, | |||
##6 ul 25 mM dNTP, | |||
##12 ul 2 mM aminoallyl dUTP | |||
##9 ul Phi29 DNA polymerase (Epicentre, low concentration) | |||
##*600ul Total Volume | |||
#Add 50ul to each well and incubate overnight at 30C. (Put in at 7pm 2/26/14) | |||
#Wash 1X PBS once | |||
#Add 50ul BS(PEG)9 mix to each well and incubate @RT for 1hr (12ul BS(PEG)9 + 588ul 1X PBS) | |||
#Wash 1X PBS twice | |||
#Add 50ul 1M Tris pH 8.0 @RT for 1hr | |||
#Wash 1X PBS twice | |||
====Detection==== | |||
*To all wells add 50ul 1uM dcProbe2-Cy3 (6ul 100uM dcProbe2-Cy3 + 180ul 100% formamide + 60ul 20X SSC + 354ul H2O preheated to 75C) | |||
**Strip E6 & F6 with 80% formamide @ 37C | |||
*To E6 & F6 add 50ul 1uM dcProbe0-Cy3 (10ul 10uM dcProbe0-Cy3 + 30ul 100% formamide + 10ul 20X SSC + 50ul H2O preheated to 75C) | |||
**Strip with 80% formamide @37C | |||
*To E6 & F6 add 50ul 1uM dcProbe1-Cy3 (10ul 10uM dcProbe1-Cy3 + 30ul 100% formamide + 10ul 20X SSC + 50ul H2O preheated to 75C) | |||
*'''Images saved under 2-27-2014 folder''' | |||
===Results=== | |||
*Whole experiment summarized in powerpoint: [[File:FridayZhangMeeting_140228.pptx]] | |||
====Positive Control - Fixed Template (F9 & F10)==== | |||
*Comparing # of Fixed Template with # of 2nd Rolonies Generated | |||
**F9 used 0.5U/ul Ampligase while F10 used 1U/ul | |||
**When using MALAT1_1stRolonyFISH to count Fixed Templates, only counted large clumps of targets while single targets may not be bright enough to see | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''# Fixed Template''' | |||
| align="center" style="background:#f0f0f0;"|'''# 2nd Rolonies''' | |||
|- | |||
| F9 (0.5 U/ul)||30||5,624 | |||
|- | |||
| F10 (1 U/ul)||61||7,883 | |||
|} | |||
*Secondary Rolonies were many! Looks Great! | |||
**Here is F9 example PISA7 counted (red marks counted rolonies) image while raw tif can be found in powerpoint | |||
[[File:F9_dcProbe2_G550_Cy3_ch00_MIP_PISA7Overlay.png | 450px]] | |||
====Ampligase Concentration Comparison: 0.5U/ul vs 1U/ul==== | |||
*Looking at # of 2nd Rolonies Generated and assuming about same # of fixed template in each | |||
**C9 & F9 are 0.5U/ul | |||
**C10 & F10 are 1U/ul | |||
*No significant difference so will keep using 0.5U/ul | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Ampligase Conc''' | |||
| align="center" style="background:#f0f0f0;"|'''0.5 U/ul''' | |||
| align="center" style="background:#f0f0f0;"|'''1 U/ul''' | |||
|- | |||
| Row C||5,532||4,536 | |||
|- | |||
| Row F||5,624||7,883 | |||
|} | |||
====Positive Control - Fixed Precircularized ppMALAT1_NH2 (F7 & F8)==== | |||
*It was hard to tell if any was fixed in the first place with dcProbe2RevComp-Cy3 | |||
*2nd Rolony results seem to confirm that since there was less 2nd Rolonies than even the negative control | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''# Fixed Circle''' | |||
| align="center" style="background:#f0f0f0;"|'''# 2nd Rolonies''' | |||
|- | |||
| F7||9 (high gain)||5 | |||
|- | |||
| F8||12 (high gain)||11 | |||
|} | |||
====Positive Control - Not Fixed Precircularized ppMALAT1 (C7 & C8)==== | |||
*C8 had ~5-10X more secondary rolonies than C7 but both were way more than what we've seen before | |||
**No point actually counting since there's nothing to compare to | |||
*Example of C8 | |||
[[File:C8_dcProbe2_G550_Cy3_ch00_MIP.png | 450px]] | |||
====Negative Control (D7 & E7)==== | |||
*Saw more signal than many 2nd Rolony Ampligase tests from before so should do negative control every time in the future to get a baseline of false positives | |||
*D7: 43 | |||
*E7: 28 | |||
====Experimental Sample (E6 & F6)==== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''E6 Targets''' | |||
| align="center" style="background:#f0f0f0;"|'''E6 2nd Rolonies''' | |||
| align="center" style="background:#f0f0f0;"|'''F6 Targets''' | |||
| align="center" style="background:#f0f0f0;"|'''F6 2nd Rolonies''' | |||
|- | |||
| ACTB||6||28||6||13 | |||
|- | |||
| RAB7A||7||46||6||15 | |||
|- | |||
| MALAT1||5||246||4||145 | |||
|} | |||
*Based on the MALAT1 results it seems that trying to quantify targets with 1stRolonyFISH probes isn't very accurate | |||
*Since the 2nd Rolonies of ACTB and RAB7A are in the range of Negative control we can't confidently say any ACTB or RAB7A padlock probes produced 2nd Rolonies | |||
*It's curious that only MALAT1 seemed successful | |||
**Could be that dcProbe2-Cy3 (recently ordered from IDT to replace the old dye from Illumina) which was used to detect MALAT1 works better | |||
**Could also be MALAT1 is more abundant than the other 2, which is what was reported in Harvard's FISSEQ Science paper |
Latest revision as of 23:37, 4 March 2014
Secondary Rolony Generation[edit]
Samples: Matt:LabNotes/2014-2-21
Procedure[edit]
Anneal Padlock Probes[edit]
- 1X Ampligase Buffer
- 2uM of each padlock probe
- 0.5 U/ul Ampligase
- Incubate @ 45C for 24 hours
- Wrapped in parafilm and put in ziploc with wet towel
Fixed Template - 25U Ampligase (C9 & F9)[edit]
Component | Volume |
10X Ampligase Buffer | 10 |
200uM ppMALAT1 | 1 |
5U/ul Ampligase | 10 |
H2O | 79 |
Total | 100 |
Fixed Template - 50U Ampligase (C10 & F10)[edit]
Component | Volume |
10X Ampligase Buffer | 10 |
200uM ppMALAT1 | 1 |
5U/ul Ampligase | 20 |
H2O | 69 |
Total | 100 |
1st Rolony (E6 & F6)[edit]
Component | Volume |
10X Ampligase Buffer | 10 |
200uM ppMALAT1 | 1 |
200uM ppACTB | 1 |
200uM ppRAB7A | 1 |
5U/ul Ampligase | 10 |
H2O | 77 |
Total | 100 |
Empty Negative Control (D7 & E7)[edit]
- Not going to waste Ampligase since it's highly unlikely anything will be ligated, but will do RCA in these wells
Pre-circularized Padlock Probes (C7 & C8)[edit]
- Combined all the pre-circle ppMALAT1 made on: Matt:LabNotes/2014-2-10 into 64ul
- 75% EtOH for 10 minutes
- Wash with 1X PBS 2 times
- 0.1N HCl for 2 minutes
- Wash with 1X PBS 3 times
- Add 32ul to C7 & C8
Fixed Pre-circularized Padlock Probes (F7 & F8)[edit]
- Not going to do anything during Ampligase step
Secondary RCA[edit]
- Aspirate each well but don't wash
- F7 well was dried up
- Pre-annealing the FISSEQ_ppRCA primer for the rolling circle amplification reaction.
- 3ul of RCA primer(200 uM, GATATCGGGAAGCTGA*A*G) in 597ul of 2xSSC/30% formamide
- Preheat primer at 60C (Tm = 55C when not in 30% formamide)
- Add 50ul to each well, 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.
- 513 ul ddH2O,
- 60 ul 10x Phi29 buffer,
- 6 ul 25 mM dNTP,
- 12 ul 2 mM aminoallyl dUTP
- 9 ul Phi29 DNA polymerase (Epicentre, low concentration)
- 600ul Total Volume
- Add 50ul to each well and incubate overnight at 30C. (Put in at 7pm 2/26/14)
- Wash 1X PBS once
- Add 50ul BS(PEG)9 mix to each well and incubate @RT for 1hr (12ul BS(PEG)9 + 588ul 1X PBS)
- Wash 1X PBS twice
- Add 50ul 1M Tris pH 8.0 @RT for 1hr
- Wash 1X PBS twice
Detection[edit]
- To all wells add 50ul 1uM dcProbe2-Cy3 (6ul 100uM dcProbe2-Cy3 + 180ul 100% formamide + 60ul 20X SSC + 354ul H2O preheated to 75C)
- Strip E6 & F6 with 80% formamide @ 37C
- To E6 & F6 add 50ul 1uM dcProbe0-Cy3 (10ul 10uM dcProbe0-Cy3 + 30ul 100% formamide + 10ul 20X SSC + 50ul H2O preheated to 75C)
- Strip with 80% formamide @37C
- To E6 & F6 add 50ul 1uM dcProbe1-Cy3 (10ul 10uM dcProbe1-Cy3 + 30ul 100% formamide + 10ul 20X SSC + 50ul H2O preheated to 75C)
- Images saved under 2-27-2014 folder
Results[edit]
- Whole experiment summarized in powerpoint: File:FridayZhangMeeting 140228.pptx
Positive Control - Fixed Template (F9 & F10)[edit]
- Comparing # of Fixed Template with # of 2nd Rolonies Generated
- F9 used 0.5U/ul Ampligase while F10 used 1U/ul
- When using MALAT1_1stRolonyFISH to count Fixed Templates, only counted large clumps of targets while single targets may not be bright enough to see
' | # Fixed Template | # 2nd Rolonies |
F9 (0.5 U/ul) | 30 | 5,624 |
F10 (1 U/ul) | 61 | 7,883 |
- Secondary Rolonies were many! Looks Great!
- Here is F9 example PISA7 counted (red marks counted rolonies) image while raw tif can be found in powerpoint
File:F9 dcProbe2 G550 Cy3 ch00 MIP PISA7Overlay.png
Ampligase Concentration Comparison: 0.5U/ul vs 1U/ul[edit]
- Looking at # of 2nd Rolonies Generated and assuming about same # of fixed template in each
- C9 & F9 are 0.5U/ul
- C10 & F10 are 1U/ul
- No significant difference so will keep using 0.5U/ul
Ampligase Conc | 0.5 U/ul | 1 U/ul |
Row C | 5,532 | 4,536 |
Row F | 5,624 | 7,883 |
Positive Control - Fixed Precircularized ppMALAT1_NH2 (F7 & F8)[edit]
- It was hard to tell if any was fixed in the first place with dcProbe2RevComp-Cy3
- 2nd Rolony results seem to confirm that since there was less 2nd Rolonies than even the negative control
' | # Fixed Circle | # 2nd Rolonies |
F7 | 9 (high gain) | 5 |
F8 | 12 (high gain) | 11 |
Positive Control - Not Fixed Precircularized ppMALAT1 (C7 & C8)[edit]
- C8 had ~5-10X more secondary rolonies than C7 but both were way more than what we've seen before
- No point actually counting since there's nothing to compare to
- Example of C8
File:C8 dcProbe2 G550 Cy3 ch00 MIP.png
Negative Control (D7 & E7)[edit]
- Saw more signal than many 2nd Rolony Ampligase tests from before so should do negative control every time in the future to get a baseline of false positives
- D7: 43
- E7: 28
Experimental Sample (E6 & F6)[edit]
' | E6 Targets | E6 2nd Rolonies | F6 Targets | F6 2nd Rolonies |
ACTB | 6 | 28 | 6 | 13 |
RAB7A | 7 | 46 | 6 | 15 |
MALAT1 | 5 | 246 | 4 | 145 |
- Based on the MALAT1 results it seems that trying to quantify targets with 1stRolonyFISH probes isn't very accurate
- Since the 2nd Rolonies of ACTB and RAB7A are in the range of Negative control we can't confidently say any ACTB or RAB7A padlock probes produced 2nd Rolonies
- It's curious that only MALAT1 seemed successful
- Could be that dcProbe2-Cy3 (recently ordered from IDT to replace the old dye from Illumina) which was used to detect MALAT1 works better
- Could also be MALAT1 is more abundant than the other 2, which is what was reported in Harvard's FISSEQ Science paper