Team:MIT/miRNA Group Notebook Experiment 2 3 miRNA Experiments
Probing for miRNA in tHESC (OLD)
Made with Benchling
Project: miRNA and Repressors subgroup
Authors: Elizabeth Strand
Date: 2016-08-09
Tuesday, 8/9
Using Neon
miRNA sensor plate.PNG
Set Up
20 ul total volume
In most wells:
2 ul DNA
18 ul of cells if no siRNA
In last well:
2 ul DNA
1 ul FF4 (1 uM) only in last well
1 ul Alexa Fluor (1 uM) only in last well
16 ul cells
Wells going down:
1.
hef1a:mkate
2.
hef1a:bfp
3.
both
4.
untrans
5.
miR-9
6.
miR-451
7.
miR-21
8.
n
Experimental Well:
Probing for miRNA in tHESC w/ Estrogen 1.0
Made with Benchling
Project: miRNA and Repressors subgroup
Authors: Wangui Mbuguiro
Date: 2016-08-12
Friday, 8/12
Materials
siRNA
451 siRNA: 50 uM
DNA colors
hef1a:red: 1 ug/ul
hef1a:blue: 1 ug/ul
DNA sensors
All at ~ 1 ug/ul
Experimental Wells (Originial Plan)
1.
Untransfected
2.
Red
3.
Blue
4.
Red + Blue
5.
Sensor 9-5p
6.
Sensor 184-3p
7.
Sensor 20A-3p
8.
Sensor 21-5p
9.
Sensor 34c-5p
10.
Sensor 142-5p
11.
Sensor 182-5p
12.
Sensor 451a
13.
Sensor 451 + 50 uM siRNA
Contents
Well 1
PBS: 3 ul
Cells: 12 ul
Well 2-3 (Single Colors)
DNA: 1.5 ul
PBS: 1.5 ul
Cells: 12 ul
Well 4 (Double Color)
DNA: 0.75 each mkate + bfp
PBS: 1.5 ul
Cells: 12 ul
Wells 5-12 (Sensors):
DNA: 1.5 ul
PBS: 1.5 ul
Cells: 12 ul
Well 13 (Sensor 451 + siRNA):
DNA: 1.5 ul
**siRNA: 1.5 ul
Cells: 12 ul
Notes
Didn't do 1,2, 13, 14.
Use untransfected & red control data from receptor group
well 13 is another 451a WITHOUT siRNA
WELL 26 will not have E2 added to it. JUST 451a siRNA
Readjusting tube # since I did not do some.
Experimental Wells (What Actually Happened)
No Estrogen Added
1.
Blue
2.
Red + Blue
3.
Sensor 9-5p
4.
Sensor 18-3p
5.
Sensor 20A-3p
6.
Sensor 21-5p
7.
Sensor 34c-5p
8.
Sensor 142-5p
9.
Sensor 182
10.
Sensor 451a
11.
Sensor 451a
Estrogen added
1.
Blue
2.
Red + Blue
3.
Sensor 9-5p
4.
Sensor 184-3p
5.
Sensor 20A-3p
6.
Sensor 21-5p
7.
Sensor 34c-5p
8.
Sensor 142-5p
9.
Sensor 182
10.
Sensor 451a
11.
Sensor 451a -- 50 uM siRNA
Results.. Could not get big enough difference with estrogen induction
Also had really poor growth + contamination
Probing for miRNA in tHESC w/ Estrogen 2.0
Made with Benchling
Project: miRNA and Repressors subgroup
Authors: Elizabeth Strand
Date: 2016-09-16
Friday, 9/16
Materials
siRNA
451 siRNA: 50 uM
DNA colors
hef1a:red: 1 ug/ul
hef1a:blue: 1 ug/ul
DNA sensors
All at ~ 1 ug/ul
Experimental Wells
1.
Untransfected
2.
Red
3.
Blue
4.
Red + Blue
5.
Sensor 9-5p
6.
Sensor 18A-3p
7.
Sensor 20A-3p
8.
Sensor 21-5p
9.
Sensor 34c-5p
10.
Sensor 142-5p
11.
Sensor 200b
12.
Sensor 451a
13.
Sensor 451 + 50 uM siRNA
14-26 are the same --> ADD ESTROGEN TOMORROW
Contents
Well 1
PBS: 3 ul
Cells: 12 ul
Well 2-3 (Single Colors)
DNA: 1.5 ul
PBS: 1.5 ul
Cells: 12 ul
Well 4 (Double Color)
DNA: 0.75 each mkate + bfp
PBS: 1.5 ul
Cells: 12 ul
Wells 5-12 (Sensors):
DNA: 1.5 ul
PBS: 1.5 ul
Cells: 12 ul
Well 13 (Sensor 451 + siRNA):
DNA: 1.5 ul
**siRNA: 1.5 ul --> Add this just before transfection
Cells: 12 ul
Dilutions
Done
A | B | C | D | E | F | G | H | I | J | |
1 | 9-5p | 18a-39 | 21-5p | 34c-5p | 200b-3p | 451 | ||||
2 | Initial C (ng/ul) | 1960.80 | 1807.30 | 1846.30 | 1330.80 | 1951.20 | 2805.10 | |||
3 | Sensor to add | 5.10 | 5.53 | 5.42 | 7.51 | 5.13 | 3.56 | |||
4 | H2O to Add | 4.90 | 4.47 | 4.58 | 2.49 | 4.87 | 6.44 | |||
5 | Final C (ng/ul) | 1000.00 | 1000.00 | 1000.00 | 1000.00 | 1000.00 | 1000.00 | |||
6 | Total volume | 10.00 | 10.00 | 10.00 | 10.00 | 10.00 | 10.00 |
Table1
