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

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(3A Ligation of Mer promoter+Mer regulatory protein (pTO40) and RBS+GFP (I13500) into pSB1AT3)
(3A Ligation of Mer promoter+Mer regulatory protein (pTO40) and RBS+GFP (I13500) into pSB1AT3)
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==PCR Purification of Digested I13500 (JCH)==
==PCR Purification of Digested I13500 (JCH)==
*Nanodrop: 23.3 ng/µL
*Nanodrop: 23.3 ng/µL
-
==3A Ligation of Mer promoter+Mer regulatory protein (pTO40) and RBS+GFP (I13500) into pSB1AT3==
+
==3A Ligation of Mer promoter+Mer regulatory protein (pTO40) and RBS+GFP (I13500) into pSB1AT3 (SK, JH, BH)==
<pre>
<pre>
Part          Length          Nanodrop
Part          Length          Nanodrop
Line 37: Line 37:
I13500        740            23.3
I13500        740            23.3
</pre>
</pre>
-
*Ligation Mixtures Calculations
+
*Ligation Mixtures Calculations (see wiki Notebook 7/9)
<pre>
<pre>
Backbone: (50 ng) / (125 ng/µl) = 0.4 µl of pSB1AT3
Backbone: (50 ng) / (125 ng/µl) = 0.4 µl of pSB1AT3

Revision as of 14:32, 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 (SK, JH, BH)

Part          Length          Nanodrop
pSB1AT3       3450 bp         125 ng/µl
pT040         700             29.9
I13500        740             23.3
  • Ligation Mixtures Calculations (see wiki Notebook 7/9)
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
Positive Sample, "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

Negative Control (backbone only), "iGEM 7/23 Lig. - in 1AT3"
0.4  µl pSB1AT3
0.5  µl ligase (T4)
2.0  µl ligase buffer 10x
17.1 µl H2O
-----------
20.0 µl total

  • Two PCR tubes (+ and -) put in Thermocycler, Block A, OVERNIGHT
    • Program name "16IGEM"
    • No heated lid
    • 16 degrees Celsius, forever

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
  1. Add AHL/IPTG to Liquid Culture
  2. Wait an hour for AHL induced expression
  3. Incubate wells in wash media for 30 minutes on rocker at speed 5
  4. Centrifuge cells out of Liquid Culture media
  5. Resuspend in wash media
  6. Add cells to appropriate well
  7. Place on rocker for 1 hour at speed 5
  8. Aspirate/Pipet out media in wells
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