Difference between revisions of "Team:Michigan/Notebook/July"

 
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<strong>7/02/2015</strong>
 
<strong>7/02/2015</strong>
 
<br>
 
<br>
−
-Tested Liu Lab plasmids and our switches (G/H)
+
Tested Liu Lab plasmids and our switches (G/H)
−
<br>
+
<br><br>
−
In Vitro Translation kit salt con:
+
In Vitro Translation kit salt concentrations:
 
<br>
 
<br>
 
[Mg] = 8-12nM
 
[Mg] = 8-12nM
Line 15: Line 15:
 
[K] = 120nM
 
[K] = 120nM
 
<br><br>
 
<br><br>
 +
Concentrations of our switches:<br>
 +
*After phenol:chloroform extraction
 +
<table style="width:80%">
 +
  <tr>
 +
    <td>Switch G</td>
 +
    <td>55 ug/ml</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Trigger for Switch G (with leader)</td>
 +
    <td>100uM (just diluted from synthesis)</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Switch H</td>
 +
    <td>58.8 ug/ml</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Trigger for Switch H (with leader)</td>
 +
    <td>60 uM (just diluted frin synthesis)</td>
 +
  </tr>
 +
</table><br><br>
  
−
<strong>6/15/2015</strong>
+
Liu Lab plasmids:<br>
−
<br>
+
*After phenol:chloroform extraction<br>
−
-Made ampicillin and chloramphenicol plates
+
<table style="width:80%">
−
<br><br>
+
  <tr>
 +
    <td>Switch + GFP</td>
 +
    <td>40 ug/ml</td>
 +
  </tr>
 +
  <tr>
 +
    <td>GFP (positive control)</td>
 +
    <td>60 ug/ml</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Switch + Mcherry</td>
 +
    <td>43 ug/ml</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Mcherry (positive control)</td>
 +
    <td>52 ug/ml</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Trigger '16'</td>
 +
    <td>55 ug/ml</td>
 +
  </tr>
 +
</table><br><br>
  
−
<strong>6/16/2015</strong>
+
In-Vitro Protein Synthesis Kit:
−
<br>
+
<table style="width:80%">
−
-Plasmid transformation of plasmids we obtained using 1ul of DNA and 50uls of NEB competent cells
+
  <tr>
−
<br><br>
+
    <td>Solution A:</td>
 +
    <td>5 uls</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Solution B:</td>
 +
    <td>3.75 uls</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Supplements (RNAse Inhibitor)<br>*20units RNAse Inhibitor</td>
 +
    <td>0.5uls</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Template DNA</td>
 +
    <td>1.0 uls (depending on how many samples)</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Nuclease-free water</td>
 +
    <td>12.5 uls</td>
 +
  </tr>
 +
</table><br><br>
  
−
<strong>6/17/2015</strong>
+
1 = switch 16 (GFP) with trigger<br>
−
<br>
+
2 = switch 16 (GFP) no trigger<br>
−
-Made cultures from colonies of transformation
+
3 = switch H with no trigger<br>
−
<br><br>
+
4 = switch H with trigger<br>
 +
5 = switch 16 (Mcherry) with no trigger<br>
 +
6 = switch 16 (Mcherry) with trigger<br>
 +
7 = switch G without trigger<br>
 +
9 = switch G with trigger<br><br>
  
 +
<img src="https://static.igem.org/mediawiki/2015/0/04/July_table1.png" alt="Mountain View" style="width:750px;height:400px;"><br><br>
 +
<img src="https://static.igem.org/mediawiki/2015/a/ad/July_graph_1.png" alt="Mountain View" style="width:750px;height:400px;"><br><br>
 +
<img src="https://static.igem.org/mediawiki/2015/1/1d/July_table_2REAL.png" alt="Mountain View" style="width:750px;height:400px;"><br><br>
  
−
<strong>6/18/2015</strong>
+
<strong>7/06/2015</strong>
 
<br>
 
<br>
−
-Mini-prep of plasmids from cultures
+
-Need to pick colonies from full synthesis product to synthesize and start cloning to GFP vector
−
<br>
+
−
-Sent for sequencing with double primers
+
 
<br><br>
 
<br><br>
  
−
<strong>6/22/2015</strong>
+
<strong>7/07/2015</strong>
 
<br>
 
<br>
−
-Miniprep of Liu lab plasmids. Stored in yellow rack in the freezer
+
-Work to do: miniprep synthesis cultures
−
<br>  
+
<br>
−
-Sequence verified
+
-PCR
 +
<br>
 +
-Submit samples for sequencing
 +
<br>
 +
-Make ampicillin plates
 
<br><br>
 
<br><br>
  
−
<strong>6/28/2015</strong>
+
<strong>7/08/2015</strong>
 
<br>
 
<br>
−
-Plasmid transformations of both switches from synthesis order (G flip and H flip)
+
-Digest synthesis with E/P to insert into iGEM vector (psb1C3)
 
<br>
 
<br>
−
-Diluted synthesis with 50uls of ultra pure water, so we should have a concentration of 80ug/ul
+
-Cultures didn’t grow, we need more Media
 
<br>
 
<br>
−
-pIDTBlue vector from IDT has amp resistance
+
 
 +
-tested Liu lab plasmid switches with and without their respective triggers
 +
<br><br>
 +
100uM trigger
 
<br>
 
<br>
−
-NOTE: one of the unused Amp plate had contamination (undesired colony), once we get the plasmid transformation colonies, we should transfer them to new Amp plates. Need to get new Amp
+
58 ng/ul switch
 
<br><br>
 
<br><br>
  
−
<strong>6/29/2015</strong>
+
 
 +
<strong>7/10/2015</strong>
 
<br>
 
<br>
−
-pick colonies from plasmid transformations to miniprep
+
Tested our switches(G/H) with and without thrombin
−
<br>  
+
<br>
−
-NOTE: New Amp is on the fridge
+
Also tested them with their two different triggers (with/without leader)
 +
<br><br>
 +
33nM linear switch
 +
<br><br>
 +
5uM trigger<br>
 +
7.5 uM DNA aptamer<br>
 +
(incubated trigger with aptamer for 15 minutes)<br><br>
 +
 
 +
11. 25 uM thrombin<br><br>
 +
 
 +
Try next:<br>
 +
Pick one trigger switch combo<br>
 +
58 ng/ul switch plasmid (same as Exp 1)<br>
 +
100uM trigger (same as Exp 1)<br>
 +
100uM DNA aptamer (same concentration as trigger)<br>
 +
Incubate trigger + thrombin overnight<br><br>
 +
 
 +
different concentrations of Thrombin.  Jenn recommends: 5uM, 11.25uM (same as Exp 2), 25uM, 100uM, 150uM<br>
 +
choose 1 switch plasmid<br>
 +
choose trigger with leader<br><br>
 +
 
 +
Order new aptamer+ ”junk” oligos - Jenn will email<br><br>
 +
 
 +
1 = switch G + Thrombin + Aptamer + Trigger<br>
 +
2 = switch G + Thrombin + Aptamer + Trigger + leader<br>
 +
3 = switch H + Thrombin + Aptamer + Trigger<br>
 +
4 = switch H + Thrombin + Aptamer + Trigger + leader<br>
 +
5 = switch G + Aptamer + Trigger<br>
 +
6 = switch G + Aptamer + Trigger + leader<br>
 +
7 = switch H + Aptamer + Trigger<br>
 +
8 = switch H + Aptamer + Trigger + leader<br><br>
 +
<img src="https://static.igem.org/mediawiki/2015/c/cc/July_table_3.png" alt="Mountain View" style="width:700px;height:350px;"><br><br>
 +
<img src="https://static.igem.org/mediawiki/2015/6/62/Julyfinalgraph.png" alt="Mountain View" style="width:750px;height:400px;"><br><br>
 +
<img src="https://static.igem.org/mediawiki/2015/e/ed/July_final_table.png" alt="Mountain View" style="width:750px;height:500px;"><br><br>
 
<br><br>
 
<br><br>
  
−
<strong>6/30/2015</strong>
+
<strong>7/18/2015</strong>
 +
<br>
 +
tested kit with thrombin using a 1:1 ratio of trigger and aptamer<br>
 +
- Incubated aptamer and trigger for 7 hours
 +
<br>
 +
Thrombin Induced Reaction
 +
<table style="width:80%">
 +
  <tr>
 +
    <td>Solution A:</td>
 +
    <td>5 uls</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Solution B:</td>
 +
    <td>3.75 uls</td>
 +
  </tr>
 +
  <tr>
 +
    <td>RNase</td>
 +
    <td>0.5uls (20 units)</td>
 +
  </tr>
 +
  <tr>
 +
    <td>switch</td>
 +
    <td>1.0 ul</td>
 +
  </tr>
 +
  <tr>
 +
    <td>trigger-aptamer</td>
 +
    <td>2.00 uls (8uM)</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Thrombin</td>
 +
    <td>0.785 ul (11.25uM)</td>
 +
  </tr>
 +
  <tr>
 +
    <td>DI H<sub>2</sub>O</td>
 +
    <td>1.965 uls</td>
 +
  <tr>
 +
    <td>Total</td>
 +
    <td>15.00 uls (20% more than 12.5 uls)</td>
 +
  </tr>
 +
</table><br>
 +
● 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 <br><br>
 +
Negative Control Reaction: (without Thrombin)
 +
<table style="width:80%">
 +
  <tr>
 +
    <td>Solution A:</td>
 +
    <td>5.00 uls</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Solution B:</td>
 +
    <td>3.75 uls</td>
 +
  </tr>
 +
  <tr>
 +
    <td>RNase</td>
 +
    <td>0.50 uls</td>
 +
  </tr>
 +
  <tr>
 +
    <td>switch</td>
 +
    <td>1.00 ul</td>
 +
  </tr>
 +
  <tr>
 +
    <td>trigger-aptamer</td>
 +
    <td>2.00 uls</td>
 +
  </tr>
 +
  <tr>
 +
    <td>DI H<sub>2</sub>O</td>
 +
    <td>2.75 uls</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Total</td>
 +
    <td>15.00 uls</td>
 +
</table><br>
 +
We wanted to keep the final volumes the same
 
<br>
 
<br>
−
-Miniprep G and H switch flip
 
 
<br><br>
 
<br><br>
−
 
  
  
 
</p>
 
</p>
  
−
</html>
+
 
  
  

Latest revision as of 20:50, 24 October 2015

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