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=Test Diffusion of PA Gel Formulations= | |||
*Using [[Matt:LabNotes/2016-3-31|PKP2 Beads]] as targets | |||
*CA12kNov2014 V4 Padlock Probes | |||
==Experiment Plan== | |||
*Goal: Test whether padlock probes and polymerases can diffuse through ~400um polyacrylamide hydrogel to generate rolonies | |||
*Dish1: Positive control: mix beads into hydrogel solution before polymerization | |||
**This way some beads will be close to the surface, minimizing distance molecules need to diffuse | |||
**Can't do it without hydrogel matrix because the beads and rolonies need some sort of substrate | |||
*Dish2: 10% Polyacrylamide Gel (199:1 Acrylamide:Bis-acrylamide) | |||
**Beads are underneath gel, being held in place by magnets | |||
*Dish3: 10% Polyacrylamide (199:1) + 4% PFA Gel | |||
**Beads are underneath gel, being held in place by magnets | |||
*Do padlock probe capture and then RCA to generate rolonies in hydrogel matrix | |||
==Protocol== | |||
===Day 1=== | |||
#Prepare hydrogel mix | |||
##Positive Control: [[Matt:LabNotes/2016-2-9#Experiment|Standard hydrogel]] + 2ul of PKP2 beads | |||
##10% PA Gel: [[Matt:LabNotes/2016-2-9#Experiment|Standard hydrogel]] | |||
##10% PA Gel + 4% PFA: [[Matt:LabNotes/2016-2-9#Experiment|Standard hydrogel]] + 4% PFA | |||
#Attach 3 Vectabond + Bind-silane treated coverslips to 3 culture dishes | |||
#Add 3ul PKP2 beads diluted in 50ul H2O to coverslips right on top of 2 magnets | |||
#*Pipette away extra H2O leaving only dried beads | |||
#*[[File:20160411_magnetbeads_culturedish.jpg|250px]] | |||
#Inject ~70ul of 200ul hydrogel mix into each dish (underneath the plastic insert) | |||
#Vacuum seal bag and then pump with Argon gas | |||
#Let set at RT for 30min | |||
#Wash with nf-H2O twice | #Wash with nf-H2O twice | ||
#Prepare Ampligase mix on ice | #Prepare Ampligase mix on ice | ||
#*Preheat H2O + Padlock probes to 85C and then snap cool before adding Ampligase buffer and enzyme | #*Preheat H2O + Padlock probes to 85C and then snap cool before adding Ampligase buffer and enzyme | ||
*Combine B11 and B12: 100ul at ~200nM | *Combine B11 and B12: 100ul at ~200nM | ||
**B12 looked to have some very fine PA gel still in there (not filtered enough?) | |||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Component''' | | align="center" style="background:#f0f0f0;"|'''Component''' | ||
Line 21: | Line 49: | ||
#Add mix to sample and incubate for 30min at 37C | #Add mix to sample and incubate for 30min at 37C | ||
#Move sample to 60C and oven slowly decreases to 55C and held for ~24hr | #Move sample to 60C and oven slowly decreases to 55C and held for ~24hr | ||
===Day | ===Day 2=== | ||
#Wash with 1X PBS once | #Wash with 1X PBS once | ||
#Add 1uM FISSEQ_ppRCA (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 55C | #Add 1uM FISSEQ_ppRCA (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 55C | ||
Line 45: | Line 72: | ||
| Total||200 | | Total||200 | ||
|} | |} | ||
-- | ===Day 3=== | ||
*Dish1 showed signs of leaking but the gel was not dried out and there was still some liquid on top | |||
**Later confirmed to definitely be leaking | |||
#Wash with 1X PBS twice | |||
#Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) for 1hr at RT | |||
#Wash with 1X PBS twice | |||
#Add 1M Tris ph8.0 for 30min at RT | |||
==Imaging== | |||
===Dish 1: Positive Control=== | |||
*Beads well dispersed among many z layers | |||
**No z-slice has them all in focus | |||
====Hybridize FISGA_Adpt Cy3==== | |||
#Preheat 600ul 0.5uM FISGA_Adpt_Cy3 in 30% formamide 2X SSC to 75C | |||
#Add 200ul to each dish and let sit for 10min at RT | |||
#Wash twice with 2X SSC | |||
*Position 1 | |||
**Initially thought good, there is signal as expected | |||
[[File:MAX_Dish1_PosCtrl_FISGA_Adpt_Pos1.jpg|450px]] | |||
*Position 2 | |||
**See some spots but they overlap with bead position (and not the beads closest to the top surface) | |||
**Not consistent with position 1 results | |||
[[File:Dish1_PosCtrl_FISGA_Adpt_Pos2_z07_ch01.jpg|450px]][[File:MAX_Dish1_PosCtrl_FISGA_Adpt_Pos2.jpg|450px]] | |||
====Strip==== | |||
#Preheat 80% formamide 2X SSC to 75C | |||
#Add 200ul to Dish1 (PosCtrl) and let sit for 15min at RT | |||
#Wash twice with 1X PBS | |||
#Add 500ul 2x SSC to image | |||
*Bumped dish, lost position but found again | |||
*Position 1 (Cy3) | |||
**Not good sign, signal is still there... even though it looks like less | |||
[[File:MAX_Dish1_PosCtrl_Stripped_Cy3_Pos1.jpg|450px]] | |||
*Position 1 (Cy5) | |||
[[File:MAX_Dish1_PosCtrl_Stripped_Cy5_Pos1.jpg|450px]] | |||
====Hybridize Cycle5 Cy3 & Cy5==== | |||
*Only Cy5 should show signal | |||
*Position 1 (Cy3) | |||
[[File:MAX_Dish1_PosCtrl_Cycle5_Cy3_Pos1.jpg|450px]] | |||
*Position 1 (Cy5) | |||
**Good there's signal when there wasn't before! | |||
[[File:MAX_Dish1_PosCtrl_Cycle5_Cy5_Pos1.jpg|450px]] | |||
===Dish 2: 10% PA=== | |||
*Beads are clearly in focus in single z-plane | |||
**High density where magnet was | |||
[[File:Dish2_10%25PA_z07_ch01.jpg|450px]] | |||
*Hybridized FISGA_Adpt_Cy3 but only a few fluorescent spots, not sure if rolonies or beads... | |||
**Turned up brightness in ImageJ and can see dim beads | |||
**However if the few bright spots are rolonies I expected to see more | |||
[[File:MAX_Dish2_10%25PA.jpg|450px]] | |||
===Dish 3: 10% PA 4% PFA=== | |||
*Beads are clearly in focus in single z-plane | |||
**High density where magnet was | |||
[[File:Dish3_10%25PA_4%25PFA_z07_ch01.jpg|450px]] | |||
*Hybridized FISGA_Adpt_Cy3 but No Fluorescence!! | |||
**Turned up brightness in ImageJ and only signal is from beads | |||
[[File:MAX_Dish3_10%25PA_4%25PFA.jpg|450px]] | |||
=Order PKP2 Padlock Probe from IDT= | |||
*To run these tests without wasting time/$/effort on padlock probe production from CA12k_Nov2014 V4 | |||
*Order single padlock probe from IDT | |||
PKP2 PKP2_3 chr12:32802524-32802525 GAGATGGCTGTCTTTTTCACACTTGG 63 GTCACCAACATGCAGCATCTTTC 63 2.719207 W GAGATGGCTGTCTTTTTCACACTTGGGTCACCAACATGCAGCATCTTTC B02203 | |||
*PKP2_controlPP: /5Phos/CCAAGTGTGAAAAAGACAGCCATCTCCTTCAGCTTCCCGATATCCGACGGTAGTGTCGAAAGATGCTGCATGTTGGTGAC |
Latest revision as of 21:01, 22 April 2016
Test Diffusion of PA Gel Formulations[edit]
- Using PKP2 Beads as targets
- CA12kNov2014 V4 Padlock Probes
Experiment Plan[edit]
- Goal: Test whether padlock probes and polymerases can diffuse through ~400um polyacrylamide hydrogel to generate rolonies
- Dish1: Positive control: mix beads into hydrogel solution before polymerization
- This way some beads will be close to the surface, minimizing distance molecules need to diffuse
- Can't do it without hydrogel matrix because the beads and rolonies need some sort of substrate
- Dish2: 10% Polyacrylamide Gel (199:1 Acrylamide:Bis-acrylamide)
- Beads are underneath gel, being held in place by magnets
- Dish3: 10% Polyacrylamide (199:1) + 4% PFA Gel
- Beads are underneath gel, being held in place by magnets
- Do padlock probe capture and then RCA to generate rolonies in hydrogel matrix
Protocol[edit]
Day 1[edit]
- Prepare hydrogel mix
- Positive Control: Standard hydrogel + 2ul of PKP2 beads
- 10% PA Gel: Standard hydrogel
- 10% PA Gel + 4% PFA: Standard hydrogel + 4% PFA
- Attach 3 Vectabond + Bind-silane treated coverslips to 3 culture dishes
- Add 3ul PKP2 beads diluted in 50ul H2O to coverslips right on top of 2 magnets
- Pipette away extra H2O leaving only dried beads
- File:20160411 magnetbeads culturedish.jpg
- Inject ~70ul of 200ul hydrogel mix into each dish (underneath the plastic insert)
- Vacuum seal bag and then pump with Argon gas
- Let set at RT for 30min
- Wash with nf-H2O twice
- Prepare Ampligase mix on ice
- Preheat H2O + Padlock probes to 85C and then snap cool before adding Ampligase buffer and enzyme
- Combine B11 and B12: 100ul at ~200nM
- B12 looked to have some very fine PA gel still in there (not filtered enough?)
Component | Volume |
DEPC-H2O | 47 |
Ampligase Buffer | 10 |
Batch 11 & 12 Padlock Probes ~200nM]] | 33 |
Ampligase | 10 |
Total | 100 |
- Add mix to sample and incubate for 30min at 37C
- Move sample to 60C and oven slowly decreases to 55C and held for ~24hr
Day 2[edit]
- Wash with 1X PBS once
- Add 1uM FISSEQ_ppRCA (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 55C
- Preheated to 70C before adding
- Wash with 2X SSC once, 1X SSC once, and 1X PBS once
- Add RCA mix and incubate 15hr at 30C
Component | Volume |
H2O | 174 |
10X Phi29 Buffer | 20 |
25mM dNTP | 2 |
4mM aa-dUTP | 2 |
Phi 29 (low conc) | 2 |
Total | 200 |
Day 3[edit]
- Dish1 showed signs of leaking but the gel was not dried out and there was still some liquid on top
- Later confirmed to definitely be leaking
- Wash with 1X PBS twice
- Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) for 1hr at RT
- Wash with 1X PBS twice
- Add 1M Tris ph8.0 for 30min at RT
Imaging[edit]
Dish 1: Positive Control[edit]
- Beads well dispersed among many z layers
- No z-slice has them all in focus
Hybridize FISGA_Adpt Cy3[edit]
- Preheat 600ul 0.5uM FISGA_Adpt_Cy3 in 30% formamide 2X SSC to 75C
- Add 200ul to each dish and let sit for 10min at RT
- Wash twice with 2X SSC
- Position 1
- Initially thought good, there is signal as expected
File:MAX Dish1 PosCtrl FISGA Adpt Pos1.jpg
- Position 2
- See some spots but they overlap with bead position (and not the beads closest to the top surface)
- Not consistent with position 1 results
File:Dish1 PosCtrl FISGA Adpt Pos2 z07 ch01.jpgFile:MAX Dish1 PosCtrl FISGA Adpt Pos2.jpg
Strip[edit]
- Preheat 80% formamide 2X SSC to 75C
- Add 200ul to Dish1 (PosCtrl) and let sit for 15min at RT
- Wash twice with 1X PBS
- Add 500ul 2x SSC to image
- Bumped dish, lost position but found again
- Position 1 (Cy3)
- Not good sign, signal is still there... even though it looks like less
File:MAX Dish1 PosCtrl Stripped Cy3 Pos1.jpg
- Position 1 (Cy5)
File:MAX Dish1 PosCtrl Stripped Cy5 Pos1.jpg
Hybridize Cycle5 Cy3 & Cy5[edit]
- Only Cy5 should show signal
- Position 1 (Cy3)
File:MAX Dish1 PosCtrl Cycle5 Cy3 Pos1.jpg
- Position 1 (Cy5)
- Good there's signal when there wasn't before!
File:MAX Dish1 PosCtrl Cycle5 Cy5 Pos1.jpg
Dish 2: 10% PA[edit]
- Beads are clearly in focus in single z-plane
- High density where magnet was
- Hybridized FISGA_Adpt_Cy3 but only a few fluorescent spots, not sure if rolonies or beads...
- Turned up brightness in ImageJ and can see dim beads
- However if the few bright spots are rolonies I expected to see more
Dish 3: 10% PA 4% PFA[edit]
- Beads are clearly in focus in single z-plane
- High density where magnet was
File:Dish3 10%PA 4%PFA z07 ch01.jpg
- Hybridized FISGA_Adpt_Cy3 but No Fluorescence!!
- Turned up brightness in ImageJ and only signal is from beads
File:MAX Dish3 10%PA 4%PFA.jpg
Order PKP2 Padlock Probe from IDT[edit]
- To run these tests without wasting time/$/effort on padlock probe production from CA12k_Nov2014 V4
- Order single padlock probe from IDT
PKP2 PKP2_3 chr12:32802524-32802525 GAGATGGCTGTCTTTTTCACACTTGG 63 GTCACCAACATGCAGCATCTTTC 63 2.719207 W GAGATGGCTGTCTTTTTCACACTTGGGTCACCAACATGCAGCATCTTTC B02203
- PKP2_controlPP: /5Phos/CCAAGTGTGAAAAAGACAGCCATCTCCTTCAGCTTCCCGATATCCGACGGTAGTGTCGAAAGATGCTGCATGTTGGTGAC