Differences

This shows you the differences between two versions of the page.

Link to this comparison view

Both sides previous revision Previous revision
current_recipes_for_cnt_conjugation [2012/05/02 11:27]
landon
current_recipes_for_cnt_conjugation [2012/05/02 13:16] (current)
will
Line 6: Line 6:
   * 100x50 Bath Dish (x4) - For various baths   * 100x50 Bath Dish (x4) - For various baths
   * 1.5 mL Microtube (x2) - For pyrene-linker and protein soaks   * 1.5 mL Microtube (x2) - For pyrene-linker and protein soaks
-  * 50 mL Plastic Falcon Tubes (x2) - For mixing solutions+  * 50 mL Plastic Falcon Tubes (x3) - For mixing solutions
   * 100 mL Beaker (x2) - For holding Ethanol and diH2O   * 100 mL Beaker (x2) - For holding Ethanol and diH2O
  
Line 15: Line 15:
 Solutions: Solutions:
   * 20mM Na2HPO4 (Sodium Phosphate) (100 mL minimum)   * 20mM Na2HPO4 (Sodium Phosphate) (100 mL minimum)
-  * 0.1% Tween-20 in Ethanol (200 mL minimum)+  * 0.1% Tween-20 in Ethanol (150 mL minimum)
   * 1mM Pyrene Maleimide in Ethanol (1 mL minimum)   * 1mM Pyrene Maleimide in Ethanol (1 mL minimum)
   * 10mM KCl (Potassium Chloride) (50 mL minimum)   * 10mM KCl (Potassium Chloride) (50 mL minimum)
-  * diH2O (200mL minimum)+  * diH2O (250mL minimum)
 It is recommended to use concentrated stock solution to prepare the solutions listed above directly before performing the experiment. It is recommended to use concentrated stock solution to prepare the solutions listed above directly before performing the experiment.
  
Line 27: Line 27:
   - While the chip is shaking in the 0.05% Tween+ethanol,​ 20mM Na2HPO4 solution, prepare a 100 mL diH2O water bath in a 100x50 Bath Dish. After soaking for 10 minutes in the 0.05% Tween+ethanol,​ 20mM Na2HPO4 solution, transfer the chip to the water bath and place on the shaker for 5 minutes at 110 RPM.    - While the chip is shaking in the 0.05% Tween+ethanol,​ 20mM Na2HPO4 solution, prepare a 100 mL diH2O water bath in a 100x50 Bath Dish. After soaking for 10 minutes in the 0.05% Tween+ethanol,​ 20mM Na2HPO4 solution, transfer the chip to the water bath and place on the shaker for 5 minutes at 110 RPM. 
   - While the chip is shaking in the water bath, prepare a 0.5 mL - 1.0 mL protein solution (making sure to keep it appropriately buffered, 20mM Na2HPO4 works for a variety of proteins) of the desired concentration in a 1.5 mL Microtube. The [[http://​dx.doi.org/​10.1126/​science.1214824|2012 Science paper]] reports using a 50uM concentration,​ however lower concentrations (~100 nM) are more typical for protein experiments. After 5 minutes in the shaking water bath, the chip should be quickly transferred to the Microtube and sealed in. Make sure the face of the chip is orientated up and that the chip is completely submerged in the protein solution. The chip should soak in the protein solution at room temperature for 1 hour.    - While the chip is shaking in the water bath, prepare a 0.5 mL - 1.0 mL protein solution (making sure to keep it appropriately buffered, 20mM Na2HPO4 works for a variety of proteins) of the desired concentration in a 1.5 mL Microtube. The [[http://​dx.doi.org/​10.1126/​science.1214824|2012 Science paper]] reports using a 50uM concentration,​ however lower concentrations (~100 nM) are more typical for protein experiments. After 5 minutes in the shaking water bath, the chip should be quickly transferred to the Microtube and sealed in. Make sure the face of the chip is orientated up and that the chip is completely submerged in the protein solution. The chip should soak in the protein solution at room temperature for 1 hour. 
-  - While the chip is soaking in the protein solution, prepare a 100 mL solution of 5mM KCl, 10mM Na2HPO4, 0.05% Tween+ethanol. It is recommended that a 10mM KCl, 20mM Na2HPO4 solution is prepared in a 50 mL Falcon Tube and a 0.01% Tween+ethanol ​solution is prepared in another 50 mL Falcon Tube and that both are added to the 100x50 Bath Dish right before use. After soaking for 1 hour in the protein solution, quickly transfer the chip to the 100 mL solution of 5mM KCl, 10mM Na2HPO4, 0.05% Tween+ethanol ​and set on the shaker at 110 RPM for 30 minutes+  - While the chip is soaking in the protein solution, prepare a 100 mL solution of 5mM KCl, 10mM Na2HPO4, 0.05% Tween+diH2O. It is recommended that a 10mM KCl, 20mM Na2HPO4 solution is prepared in a 50 mL Falcon Tube and a 0.01% Tween+diH2O solution is prepared in another 50 mL Falcon Tube and that both are added to the 100x50 Bath Dish right before use. After soaking for 1 hour in the protein solution, quickly transfer the chip to the 100 mL solution of 5mM KCl, 10mM Na2HPO4, 0.05% Tween+diH2O and set on the shaker at 110 RPM for 30 minutes.
-    * The [[http://​dx.doi.org/​10.1126/​science.1214824|2012 Science paper]] reports using 5mM KCl, 10mM Na2HPO4, 0.05% Tween in DI water during this step (no ethanol).+
   - While the chip is shaking in the 100 mL solution of 5mM KCl, 10mM Na2HPO4, 0.05% Tween+ethanol,​ prepare a 100 mL diH2O bath. After shaking for 30 minutes in the 100 mL solution of 5mM KCl, 10mM Na2HPO4, 0.05% Tween+ethanol,​ quickly transfer the chip to the water bath and set on the shaker at 110 RPM for 5 minutes.   - While the chip is shaking in the 100 mL solution of 5mM KCl, 10mM Na2HPO4, 0.05% Tween+ethanol,​ prepare a 100 mL diH2O bath. After shaking for 30 minutes in the 100 mL solution of 5mM KCl, 10mM Na2HPO4, 0.05% Tween+ethanol,​ quickly transfer the chip to the water bath and set on the shaker at 110 RPM for 5 minutes.
   - After shaking in the water bath for 5 minutes, remove the chip and carefully blow dry with nitrogen. The chip has now undergone the full protocol and should have protein bound to the nanotube through a pyrene maleimide linker molecule. ​   - After shaking in the water bath for 5 minutes, remove the chip and carefully blow dry with nitrogen. The chip has now undergone the full protocol and should have protein bound to the nanotube through a pyrene maleimide linker molecule. ​
 +    - [[http://​dx.doi.org/​10.1126/​science.1214824|2012 Science paper]]
  
 __Condensed Protocol__: __Condensed Protocol__:
Line 38: Line 38:
   - 5 min shake in diH2O, 110 RPM   - 5 min shake in diH2O, 110 RPM
   - 60 min protein soak, no agitation   - 60 min protein soak, no agitation
-  - 30 min shake in a 50-50 ratio of 5mM KCl+10mM Na2HPO4 in diH2O and 0.05% Tween-20 in ethanol, 110 RPM +  - 30 min shake in a 50-50 ratio of 5mM KCl+10mM Na2HPO4 in diH2O and 0.05% Tween-20 in diH2O, 110 RPM
-    * The [[http://​dx.doi.org/​10.1126/​science.1214824|2012 Science paper]] reports using 5mM KCl, 10mM Na2HPO4, 0.05% Tween in DI water during this step (no ethanol).+
   - 5 min shake in diH2O, 110 RPM   - 5 min shake in diH2O, 110 RPM
   - Blow dry with nitrogen   - Blow dry with nitrogen

QR Code
QR Code current_recipes_for_cnt_conjugation (generated for current page)