Revision as of 19:30, 19 November 2013 by >Alan6017518
Optimum Probe# Test
- 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
- 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)
MALAT1 Padlock Probes Concentrations to test
Protocol
- In this experiment we will be using the Mattek dish containing primary rolonies made on Oct 23rd dish #2
Reagents
80% Formamide in 2X SSC
100ul MALAT1 padlock probes in 2X ssc in various concentration
- Original concentration 100uM, perform serial dilution
Stock Tube
|
Stock Volume
|
SSC
|
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
Apparatus
- warm up incubator to 50C for incubation
- warm up tube heater to 80C for pre-heating padlock probes
Stripping
- Heat 80% formaide at 80C, add to mattek dish
- Incubate for 10 mins wash with H2O twice
Annealing
- Pre-heat padlock probes at 80C for 5 mins
- Add to cells at RT, and let it cool down over 5min
Imaging