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

Revision as of 20:38, 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

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

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

</html>

@@ Line 164: / Line 164: @@
 = switch H + Aptamer + Trigger<br>
 = 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:750px;height:350px;"><br><br>
+<img src="https://static.igem.org/mediawiki/2015/c/cc/July_table_3.png" alt="Mountain View" style="width:650px;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:400px;"><br><br>