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
- 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)
- 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]
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