Optimum Probe# Test[edit]
- In this experiment we are trying to find the concentration of probes that works best for capturing rolony targets. From previous experiments we know that a concentration of 0.6uM with a volume of 100ul works.
- A test of 5 different concentrations will be perform and image analysis using PISA will be performed to count the number of target being captured
MALAT1 Padlock Probes Concentrations to test[edit]
- In this experiment we will be using the 96well plate containing primary rolonies made on cy5 primary rolony detection probe
Reagents[edit]
80% Formamide in 2X SSC
100ul MALAT1 padlock probes in 2X ssc in various concentration (ppMALAT1)
- Original concentration 100uM, perform serial dilution
Stock Tube
|
Stock Volume
|
2XSSC
|
Dilution
|
Final concentration nM
|
100000
|
7.5
|
142.5
|
0.05
|
5000
|
5000
|
3
|
147
|
0.02
|
100
|
100
|
15
|
135
|
0.1
|
10
|
10
|
15
|
135
|
0.1
|
1
|
1
|
15
|
135
|
0.1
|
0.1
|
MALAT1 padlock probes detection
- Dilute 110ul 10uM DC_Probe_2_Cy3 with 440ul 2X SSC
- Shifted FISSEQ_2ndRCAprimerDye : 2nd Rolony from all padlock probes GGATATCGGGAAGCTGAA/3ATTO488N/
Apparatus[edit]
- warm up incubator to 50C for incubation
- warm up tube heater to 80C for pre-heating padlock probes
Protocol[edit]
Image and Count primary rolonies[edit]
- Pick 6 positions and image the rolonies (Cy5)
Stripping[edit]
- Heat 80% formaide at 80C, add ~50ul to 96 well plate
- Incubate for 15 mins wash 3 times with 200ul pbs
Annealing Padlock Probes[edit]
- Pre-heat padlock probes at 80C for 5 mins
- aspirate
- Add 50ul to cells at RT
- transfer to 50C for 15 mins
- aspirate and wash with pbs 3x
Annealing Universal Padlock Detection Probes[edit]
- Preheat detection probes at 65C for 5 mins (2uM)
- Add 100ul to cells at RT incubate for 15 mins
- Wash with H2O twice and add 100ul 2x SSC
Imaging[edit]
- 10-15um, 10 z stack image, lower limit begin go up till upper limit end
- resolution (2kx2k)
- 16bit,600Hz
- when done save file (experiment_dish(mattekdate)_step#_dyeprobe_channel)
Protocol for stripping efficiency test[edit]