Team:MIT/miRNA Group Notebook Experiment 2 3 siRNA Experiments
Testing how little siRNA I can use
Made with Benchling
Project: miRNA and Repressors subgroup
Authors: Wangui Mbuguiro
Date: 2016-09-03
Saturday, 9/3
Questions being answered:
Testing minimum siRNA for saturation
1.
What's lowest amount of siRNA I can use
2.
Is my midi for FF4 ok? --> Using 451 just in case
3.
Do I get same results in tHESC?
Materials
Alexa Fluor 488: 1 uM and 10 uM
451 siRNA: 1 uM, 10 uM, 50 uM, 100 uM
FF4 siRNA: 1 uM
hef1a:red: 1 ug/ul
hef1a:blue: 1 ug/ul
451 Sensor: 1 ug/ul
FF4 Sensor: 1 ug/ul
Experimental Wells
1.
Untransfected
2.
Red
3.
Blue
4.
Red + Blue
5.
10 uM Alexa alone
6.
0 uM 451 + Alexa
7.
1 uM 451 + Alexa
8.
10 uM siRNA 451 + Alexa
9.
50 uM siRNA 451
10.
100 uM siRNA 451
11.
0 uM FF4
12.
1 uM --> 1 ul FF4 + Alexa
Contents
Well 1
PBS: 3 ul
Cells: 12 ul
Well 2-4
DNA: 1.5 ul color
PBS: 1.5 ul
Cells: 12 ul
Well 5
PBS: 1.5 ul
siRNA: 1.5 ul Alexa 10 uM
Cells: 12 ul
Wells 6-10
DNA: 1.5 ul 451 sensor
siRNA: 0.75 ul each 451 + Alexa (0, 1, 10, 50, 100)
Cells: 12 ul
Wells 11:
DNA: 1.5 ul FF4 sensor
PBS: 1.5 ul
Cells: 12 ul
Well 12:
DNA: 1.5 ul FF4 sensor
siRNA: .75 ul each FF4 + Alexa 1 uM
Cells: 12 ul
Testing miRNA Target Sites using siRNA
Made with Benchling
Project: miRNA and Repressors subgroup
Authors: Wangui Mbuguiro
Date: 2016-08-11
Thursday, 8/11
Lingering questions: if we are using a the Alexa Fluor.. how should we change set up?
0. Set Up
Inventory
siRNA at stock concentrations of: 50 uM in -80C, 1 uM in -20C
miRNA-ts choosen: miRNA-451 or FF4 (for 9, we don't have siRNA matching it)
Confirmed by miRAtlas to lack natural prescence in HEK293 cells
Purpose
With varying amounts of siRNA, how effective are miRNA-ts.
Plasmids to Transfect
clipboard_2016-08-12_11:44:42.png
Layout of Well Plate
siRNA added will probably be: 1, 2, 5, 10, 0 pmol
Need to replace 9 with FF4
Need to find out optimal amount of Lipo2K
clipboard_2016-08-11_09:27:21.png
Materials
451a sensor (100 ng/ul)
FF4 sensor (100 ng/ul)
451a siRNA (1 uM, 1 pmol/ul dilution)
FF4 siRNA (1 uM, 1 pmol/ul dilution)
hef1a: BFP (100 ng/ul)
hef1a: mKate (100 ng/ul)
0. Planning: What Ends Up In Each Well (final)
(Altering the contents of this table will automatically change the amounts to add in the next table)
A | B | C | D | E | F | G | H | I | |
1 | Well 1 | Well 2 | Well 3 | Well 4 | Well 5 | Well 6 | Well 7 | Well 8 | |
2 | What is it | hef1a BFP | hef1a:mKate | Both (2 plasmids) | Untrans | mK451 + siR451_1 | mK451 + siR451_2 | mK451 + siR451_5 | mK451 + siR451_10 |
3 | 451a sensor (ng) | 0 | 0 | 0 | 0 | 150 | 150 | 150 | 150 |
4 | FF4 sensor (ng) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
5 | 451a siRNA (pmol) | 0 | 0 | 0 | 0 | 1 | 2 | 5 | 10 |
6 | FF4 siRNA (pmol) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
7 | hef1a:BFP (ng) | 150 | 0 | 75 | 0 | 0 | 0 | 0 | 0 |
8 | hef1a:mKate (ng) | 0 | 150 | 75 | 0 | 0 | 0 | 0 | 0 |
9 | Lipo2k (uL) | 3 | 3 | 3 | 0 | 3 | 3 | 3 | 3 |
Table1
1.
Before Adding things to Wells
Set up 1 tube for each DNA+siRNA+Optimem mixture, and another large tube for the Lipo2K + Optimem mix total.
Amounts in uL
A | B | C | D | E | F | G | H | I | |
1 | Well 1 | Well 2 (in uL) | Well 3 | Well 4 | Well 5 | Well 6 | Well 7 | Well 8 | |
2 | What is it | hef1a:BFP | hef1a:mKate | Both (2 plasmids) | Untrans | mK451 + siR451_1 | mK451 + siR451_2 | mK451 + siR451_5 | mK451 + siR451_10 |
3 | 451a sensor | 0 | 0 | 0 | 0 | 3 | 3 | 3 | 3 |
4 | FF4 sensor | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
5 | 451a siRNA | 0 | 0 | 0 | 0 | 2 | 4 | 10 | 20 |
6 | FF4 siRNA | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
7 | hef1a:BFP | 3 | 0 | 1.5 | 0 | 0 | 0 | 0 | 0 |
8 | hef1a:mKate | 0 | 3 | 1.5 | 0 | 0 | 0 | 0 | 0 |
9 | Optimem | 47 | 47 | 47 | 0 | 45 | 43 | 37 | 27 |
10 | |||||||||
11 | Lipo2K | 6 | 6 | 6 | 0 | 6 | 6 | 6 | 6 |
12 | Optimem | 44 | 44 | 44 | 0 | 44 | 44 | 44 | 44 |
Table2
2. ⌚Flick tubes gently and wait 5 minutes
3. Take 25 uL of DNA+siRNA and 25 uL of Lipo2k+Optimem and combine in one tube (per well)
4. ⌚Flick tubes gently and wait 20 minutes
5. Aspirate well and add 50 uL of transfection mixture + 450 uL of fresh media
Do this for a couple of wells at a time. Try to move quickly
