Team:Michigan/Notebook/July
July
7/02/2015
Tested Liu Lab plasmids and our switches (G/H)
In Vitro Translation kit salt concentrations:
[Mg] = 8-12nM
[K] = 120nM
Concentrations of our switches:
*After phenol:chloroform extraction
| Switch G | 55 ug/ml |
| Trigger for Switch G (with leader) | 100uM (just diluted from synthesis) |
| Switch H | 58.8 ug/ml |
| Trigger for Switch H (with leader) | 60 uM (just diluted frin synthesis) |
Liu Lab plasmids:
*After phenol:chloroform extraction
| Switch + GFP | 40 ug/ml |
| GFP (positive control) | 60 ug/ml |
| Switch + Mcherry | 43 ug/ml |
| Mcherry (positive control) | 52 ug/ml |
| Trigger '16' | 55 ug/ml |
In-Vitro Protein Synthesis Kit:
| Solution A: | 5 uls |
| Solution B: | 3.75 uls |
| Supplements (RNAse Inhibitor) *20units RNAse Inhibitor |
0.5uls |
| Template DNA | 1.0 uls (depending on how many samples) |
| Nuclease-free water | 12.5 uls |
1 = switch 16 (GFP) with trigger
2 = switch 16 (GFP) no trigger
3 = switch H with no trigger
4 = switch H with trigger
5 = switch 16 (Mcherry) with no trigger
6 = switch 16 (Mcherry) with trigger
7 = switch G without trigger
9 = switch G with trigger
7/06/2015
-Need to pick colonies from full synthesis product to synthesize and start cloning to GFP vector
7/07/2015
-Work to do: miniprep synthesis cultures
-PCR
-Submit samples for sequencing
-Make ampicillin plates
7/08/2015
-Digest synthesis with E/P to insert into iGEM vector (psb1C3)
-Cultures didn’t grow, we need more Media
-tested Liu lab plasmid switches with and without their respective triggers
100uM trigger
58 ng/ul switch
7/10/2015
Tested our switches(G/H) with and without thrombin
Also tested them with their two different triggers (with/without leader)
33nM linear switch
5uM trigger
7.5 uM DNA aptamer
(incubated trigger with aptamer for 15 minutes)
11. 25 uM thrombin
Try next:
Pick one trigger switch combo
58 ng/ul switch plasmid (same as Exp 1)
100uM trigger (same as Exp 1)
100uM DNA aptamer (same concentration as trigger)
Incubate trigger + thrombin overnight
different concentrations of Thrombin. Jenn recommends: 5uM, 11.25uM (same as Exp 2), 25uM, 100uM, 150uM
choose 1 switch plasmid
choose trigger with leader
Order new aptamer+ ”junk” oligos - Jenn will email
1 = switch G + Thrombin + Aptamer + Trigger
2 = switch G + Thrombin + Aptamer + Trigger + leader
3 = switch H + Thrombin + Aptamer + Trigger
4 = switch H + Thrombin + Aptamer + Trigger + leader
5 = switch G + Aptamer + Trigger
6 = switch G + Aptamer + Trigger + leader
7 = switch H + Aptamer + Trigger
8 = switch H + Aptamer + Trigger + leader
7/18/2015
tested kit with thrombin using a 1:1 ratio of trigger and aptamer
- Incubated aptamer and trigger for 7 hours
Thrombin Induced Reaction
| Solution A: | 5 uls |
| Solution B: | 3.75 uls |
| RNase | 0.5uls (20 units) |
| switch | 1.0 ul |
| trigger-aptamer | 2.00 uls (8uM) |
| Thrombin | 0.785 ul (11.25uM) |
| DI H2O | 1.965 uls |
| Total | 15.00 uls (20% more than 12.5 uls) |
● Protocol said it was ok to go up to 20% higher of total volume, we wanted to have higher concentrations of thrombin in the solution and that made us go over the original 12.5 uls planned
Negative Control Reaction: (without Thrombin)
| Solution A: | 5.00 uls |
| Solution B: | 3.75 uls |
| RNase | 0.50 uls |
| switch | 1.00 ul |
| trigger-aptamer | 2.00 uls |
| DI H2O | 2.75 uls |
| Total | 15.00 uls |
We wanted to keep the final volumes the same