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

Mountain View

Mountain View

Mountain View

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

Mountain View

Mountain View

Mountain View



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