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

 
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<h2 style="text-align: center">July</h2>
 
<h2 style="text-align: center">July</h2>
 
<p>
 
<p>
−
<strong>6/14/2015</strong>
+
<strong>7/02/2015</strong>
 
<br>
 
<br>
−
-We got some plasmids from Dr. Allen Liu Lab, these plasmids were used in the Green paper and we plan to compare their expression to the expression of the switches we designed.
+
Tested Liu Lab plasmids and our switches (G/H)
 
<br><br>
 
<br><br>
−
 
+
In Vitro Translation kit salt concentrations:
−
<strong>6/15/2015</strong>
+
 
<br>
 
<br>
−
-Made ampicillin and chloramphenicol plates
+
[Mg] = 8-12nM
−
<br><br>
+
−
 
+
−
<strong>6/16/2015</strong>
+
 
<br>
 
<br>
−
-Plasmid transformation of plasmids we obtained using 1ul of DNA and 50uls of NEB competent cells
+
[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>6/17/2015</strong>
+
Liu Lab plasmids:<br>
−
<br>
+
*After phenol:chloroform extraction<br>
−
-Made cultures from colonies of transformation
+
<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>
  
 +
In-Vitro Protein Synthesis Kit:
 +
<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>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>6/18/2015</strong>
+
1 = switch 16 (GFP) with trigger<br>
−
<br>
+
2 = switch 16 (GFP) no trigger<br>
−
-Mini-prep of plasmids from cultures
+
3 = switch H with no trigger<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>7/06/2015</strong>
 
<br>
 
<br>
−
-Sent for sequencing with double primers
+
-Need to pick colonies from full synthesis product to synthesize and start cloning to GFP vector
 
<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>
 
<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>
  
−
<strong>6/30/2015</strong>
+
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>
 +
 
 +
<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