IGEM:MIT/2007/Notebook/2007-7-23

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Digestion of I13500)
Line 27: Line 27:
</pre>
</pre>
* Placed in 37C @ 10:20am --> Take out @ 1:20pm
* Placed in 37C @ 10:20am --> Take out @ 1:20pm
 +
 +
==PCR Purification of Digested I13500 (JCH)==
 +
*Nanodrop: 23.3 ng/µL
 +
==3A Ligation of Mer promoter+Mer regulatory protein (pTO40) and RBS+GFP (I13500) into pSB1AT3==
 +
<pre>
 +
Part          Length          Nanodrop
 +
pSB1AT3      3450 bp        125 ng/µl
 +
pT040        700            29.9
 +
I13500        740            23.3
 +
</pre>
 +
*Ligation Mixtures Calculations
 +
<pre>
 +
Backbone: (50 ng) / (125 ng/µl) = 0.4 µl of pSB1AT3
 +
Insert 1: 29.9x = (4)(700/3450)(50) --> x = 1.357 µl of Mer
 +
Insert 2: 23.3x = (4)(740/3450)(50) --> x = 1.841 µl of I13500 (cut with X and P)
 +
</pre>
 +
*Ligation Mixtures
 +
<pre>
 +
+ "iGEM 7/23 Lig + mer+I13500 in 1AT3"
 +
 +
0.4  µl pSB1AT3
 +
1.36 µl pT040 (Mer)
 +
1.84 µl I13500 (RBS+GFP)
 +
0.5  µl ligase (T4)
 +
2.0  µl ligase buffer 10x (T4)
 +
13.9 µl H2O
 +
----------
 +
20.0 µl total
 +
<pre>
==Diluted BioBrick Sequencing Primers==
==Diluted BioBrick Sequencing Primers==

Revision as of 15:26, 23 July 2007

Contents

Agenda

  1. Polystyrene Assay
    • Morning: get correct OD
    • Add AHL
    • Afternoon: Test
  2. Digest I13500
  3. Ligate 3A with I13500 and Mer in pSB1AT3 (so that, if we combine with polystyrene-binding plasmid into a single cell, can test for presence of both plasmids)
    • run overnight
  4. dilute sequencing primers
    • sequence PHIE6P
    • sequence F2620+B0034+CPX
  5. Order T7 antibody


Digestion of I13500

  • Made two mixtures
4.5 µl I13500 (Miniprep #1, 223.5 ng/µl)
1 µl   XbaI
1 µl   PstI
0.5 µl BSA
5 µl   NEB3 Buffer
38 µl  H20
-------------------------
50 µl  Total
  • Placed in 37C @ 10:20am --> Take out @ 1:20pm

PCR Purification of Digested I13500 (JCH)

  • Nanodrop: 23.3 ng/µL

3A Ligation of Mer promoter+Mer regulatory protein (pTO40) and RBS+GFP (I13500) into pSB1AT3

Part          Length          Nanodrop
pSB1AT3       3450 bp         125 ng/µl
pT040         700             29.9
I13500        740             23.3
  • Ligation Mixtures Calculations
Backbone: (50 ng) / (125 ng/µl) = 0.4 µl of pSB1AT3
Insert 1: 29.9x = (4)(700/3450)(50) --> x = 1.357 µl of Mer
Insert 2: 23.3x = (4)(740/3450)(50) --> x = 1.841 µl of I13500 (cut with X and P)
  • Ligation Mixtures
+ "iGEM 7/23 Lig + mer+I13500 in 1AT3"

0.4  µl pSB1AT3
1.36 µl pT040 (Mer)
1.84 µl I13500 (RBS+GFP)
0.5  µl ligase (T4)
2.0  µl ligase buffer 10x (T4)
13.9 µl H2O
----------
20.0 µl total
<pre>

==Diluted BioBrick Sequencing Primers==
You can find the stock eppendorfs in the unnamed -20C fridge (Jason+Barry's room).

Diluted from 100 µM stock to 32.2 µM with total volume of 1.55 mL each.

Made 1 VF2 and 1 VR for iGEM use.

==Polystyrene Assay Protocol==

* Cell Lines:
** Controls: DH5-alpha (not transformed)
** FHUA PHIE6 --> IPTG
** CPX --> AHL
*Media:
**H20
**PBS
**PBS + Tween + BSA

*Protocol
#Add AHL/IPTG to Liquid Culture
#Wait an hour for AHL induced expression
#Incubate wells in wash media for 30 minutes on rocker at speed 5
#Centrifuge cells out of Liquid Culture media
#Resuspend in wash media
#Add cells to appropriate well
#Place on rocker for 1 hour at speed 5
#Aspirate/Pipet out media in wells
Personal tools