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) | |
− | <br> | + | <br><br> |
− | In Vitro Translation kit salt | + | In Vitro Translation kit salt concentrations: |
<br> | <br> | ||
[Mg] = 8-12nM | [Mg] = 8-12nM | ||
Line 16: | Line 16: | ||
<br><br> | <br><br> | ||
− | <strong> | + | <strong>7/06/2015</strong> |
<br> | <br> | ||
− | - | + | -Need to pick colonies from full synthesis product to synthesize and start cloning to GFP vector |
<br><br> | <br><br> | ||
− | <strong> | + | <strong>7/07/2015</strong> |
<br> | <br> | ||
− | - | + | -Work to do: miniprep synthesis cultures |
+ | <br> | ||
+ | -PCR | ||
+ | <br> | ||
+ | -Submit samples for sequencing | ||
+ | <br> | ||
+ | -Make ampicillin plates | ||
<br><br> | <br><br> | ||
− | <strong> | + | <strong>7/08/2015</strong> |
<br> | <br> | ||
− | - | + | -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> | + | <strong>7/10/2015</strong> |
<br> | <br> | ||
− | + | Tested our switches(G/H) with and without thrombin | |
<br> | <br> | ||
− | + | 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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