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

Line 7: Line 7:
 
<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 16: Line 16:
 
<br><br>
 
<br><br>
  
−
<strong>6/15/2015</strong>
+
<strong>7/06/2015</strong>
 
<br>
 
<br>
−
-Made ampicillin and chloramphenicol plates
+
-Need to pick colonies from full synthesis product to synthesize and start cloning to GFP vector
 
<br><br>
 
<br><br>
  
−
<strong>6/16/2015</strong>
+
<strong>7/07/2015</strong>
 
<br>
 
<br>
−
-Plasmid transformation of plasmids we obtained using 1ul of DNA and 50uls of NEB competent cells
+
-Work to do: miniprep synthesis cultures
 +
<br>
 +
-PCR
 +
<br>
 +
-Submit samples for sequencing
 +
<br>
 +
-Make ampicillin plates
 
<br><br>
 
<br><br>
  
−
<strong>6/17/2015</strong>
+
<strong>7/08/2015</strong>
 
<br>
 
<br>
−
-Made cultures from colonies of transformation
+
-Digest synthesis with E/P to insert into iGEM vector (psb1C3)
 +
<br>
 +
-Cultures didn’t grow, we need more Media
 +
<br>
 +
 
 +
-tested Liu lab plasmid switches with and without their respective triggers
 +
<br><br>
 +
100uM trigger
 +
<br>
 +
58 ng/ul switch
 
<br><br>
 
<br><br>
  
  
−
<strong>6/18/2015</strong>
+
<strong>7/10/2015</strong>
 
<br>
 
<br>
−
-Mini-prep of plasmids from cultures
+
Tested our switches(G/H) with and without thrombin
 
<br>
 
<br>
−
-Sent for sequencing with double primers
+
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><br>
 
<br><br>
  

Revision as of 19:01, 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

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


6/22/2015
-Miniprep of Liu lab plasmids. Stored in yellow rack in the freezer
-Sequence verified

6/28/2015
-Plasmid transformations of both switches from synthesis order (G flip and H flip)
-Diluted synthesis with 50uls of ultra pure water, so we should have a concentration of 80ug/ul
-pIDTBlue vector from IDT has amp resistance
-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

6/29/2015
-pick colonies from plasmid transformations to miniprep
-NOTE: New Amp is on the fridge

6/30/2015
-Miniprep G and H switch flip



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