Team:Peking/Notebook/Protocols/PCIEG

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[[Team:Peking/Notebook|Notebook]] > [[Team:Peking/Notebook/Protocols|Protocols]] > [[Team:Peking/Notebook/Protocols/PCIEG|Protocol for ligation of insert DNA into plasmid vector DNA]]<html>
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[[Team:Peking/Notebook|Notebook]] > [[Team:Peking/Notebook/Protocols|Protocols]] > [[Team:Peking/Notebook/Protocols/PCIEG|Protocol for chemical inducible expression of GFP]]<html>
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=Protocol for Ligation of Insert DNA into Plasmid Vector DNA=
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=Protocol for Chemical Inducible Expression of GFP=
==Materials:==
==Materials:==
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* DNA sample(s) in water or TE buffer
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* 4 groups of induce solution with a concentration gradient of 10^-7, 10^-5, 10^-3, 10^-2;
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*10x ligation buffer
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* Overnight bacterial culture or bacterial colonies;
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*T4 DNA Ligase, 5 u/μl
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*ddwater
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* Phosphate Buffered Solution (PBS).
==Procedure:==
==Procedure:==
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1. Test the concentration of the DNA sample(s).
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1. Add 20 μl of the overnight bacteria l culture or pick a colony to 5ml of LB
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antibiotic medium, Incubate at 37 degree in a shaker till the OD600 value reaches 0.4-0.6.
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2. Pipet the following into a microfuge tube:
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a. Linearized vector DNA: around 100ng
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b. Insert DNA (at 3:1 molar excess over vector): variable
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c. 10x ligation buffer: 1uL
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d. T4 DNA Ligase: 1uL
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e. ddwater: Rest of volume
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'''Total volume: 10 uL'''
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3. Vortex and spin briefly to collect drops.
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4. Incubate the mixture at 16 degree for 45-60min.
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2. Add 0.5 mL of the fresh bacteria l culture and appropriate volume of inducer solution to prepare induction system with the concentration gradient of 10^-9, 10^-8, 10^-7, 10^-6, 10^-5, 10^-4, 10^-3, 10^-2.
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5. Use the ligation mixture for transformation.
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3. Place the induction system at 37 degree for 2 hours.
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4. Pellet bacteria l cells by 4 min centrifugation at 4000 rpm, discard the supernatant.
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==Tips:==
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5. Resuspend the pelleted cells in 500 μl of PBS.
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* Thoroughly mix the 10x ligation buffer before use.
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* The optima l insert/vector molar ratio is 3:1.
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6. Transfer 100 uL of bacteria l resuspention into each well of 96-well plate to test the expression of GFP by flow cytometry or Microplate Reader.
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* To minimize recircularization of the cloning vector, dephosphorylate linearized plasmid DNA with Alkaline Phospha tase(CIAP) prior to ligation. Heats inactivate the phosphatase or remove from the mixture after the dephosphorylation step.
 
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* DNA purity is an important factor for successful ligation. Plasmids should be purified using a method that will ensure isolation of high quality DNA. Use only high quality agarose and fresh electrophoresis buffers for gel-purification of DNA fragments.
 
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==Notes:==
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If desired, time sequential expression of GFP can also be tested, through verifying the incubating time of induction system at 37 degree.
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Latest revision as of 17:11, 12 October 2010




   Protocol by PKU 2010


         Notebook > Protocols > Protocol for chemical inducible expression of GFP

Contents

Protocol for Chemical Inducible Expression of GFP

Materials:

  • 4 groups of induce solution with a concentration gradient of 10^-7, 10^-5, 10^-3, 10^-2;
  • Overnight bacterial culture or bacterial colonies;
  • Phosphate Buffered Solution (PBS).


Procedure:

1. Add 20 μl of the overnight bacteria l culture or pick a colony to 5ml of LB antibiotic medium, Incubate at 37 degree in a shaker till the OD600 value reaches 0.4-0.6.

2. Add 0.5 mL of the fresh bacteria l culture and appropriate volume of inducer solution to prepare induction system with the concentration gradient of 10^-9, 10^-8, 10^-7, 10^-6, 10^-5, 10^-4, 10^-3, 10^-2.

3. Place the induction system at 37 degree for 2 hours.

4. Pellet bacteria l cells by 4 min centrifugation at 4000 rpm, discard the supernatant.

5. Resuspend the pelleted cells in 500 μl of PBS.

6. Transfer 100 uL of bacteria l resuspention into each well of 96-well plate to test the expression of GFP by flow cytometry or Microplate Reader.


Notes:

If desired, time sequential expression of GFP can also be tested, through verifying the incubating time of induction system at 37 degree.

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