Team:Chiba/Notebook/protocol

From 2009.igem.org

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__NOTOC__
== Protocols ==
== Protocols ==
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===DNA Purification===
===DNA Purification===
====Sigma prep====
====Sigma prep====
 +
 +
====Zymo DNA Cleam&Concentrator Kit====
====Zymo DNA Cleam&Concentrator Kit====
*Add 2 volume of DNA binding buffer to each volume of DNA sample, Use voltex to mix.
*Add 2 volume of DNA binding buffer to each volume of DNA sample, Use voltex to mix.
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===Agarose gel electrophoresis===
===Agarose gel electrophoresis===
 +
*Agalose Gel casting
 +
#Measure out the appropriate mass of agarose into glass bottle with the appropriate volume of TAE buffer
 +
#Microwave until the agarose is fully melted
 +
#Pour the agarose solution into the gelbox and let it cool for about 30 minutes, until the gel is solid
 +
#Remove comb
 +
 +
 +
*Running agalose gel
 +
 +
#Load 5 μL prepared 1kbp ladder
 +
#Mix DNA solution with loading dye(6x) and water
 +
#Load it into agalose gel
 +
#Run the gel at ~100 volts for 35 mins.
 +
 +
*Visualizing agarose gels
 +
#Remove gel from gel box
 +
#Soak the gel in ethidium bromide solution
 +
#Let it 30 min.
 +
#Place the gel in Trans-Illuminator and turn on UV light after make sure the door closing.
 +
#Print the picture.
 +
#Remove gel and throw in trash
 +
#Wipe down Trans-Illuminator if necessary.
 +
 +
===Digestion===
===Digestion===
 +
 +
===PCR===
===PCR===
 +
*Resuspend primer in Nuculease free water to 100 µM
 +
 +
*PCR mix
 +
#DNA template          1µL
 +
#Fwd primer            10µL (final con. 10 pM)
 +
#Rev primer            10µL
 +
#10x thermo pol buffer 10µL
 +
#dNTP mix              10µL
 +
#DNA pol.              1µL
 +
#dH20                  58µL
 +
<br>----------------------------
 +
<br>                  100µL
 +
 +
 +
*PCR cycle
 +
 +
Start: 94 °C for 5 min. (melt)
 +
cycle:          melt:            1 min.
 +
              anneal :          30 sec.
 +
cycle end: extension:72 °C for 3.5 min.
 +
25 cycles
 +
72 °C for 10 min
 +
store: keep at 6 °C forever
 +
 +
===Gel extract===
===Gel extract===
 +
 +
READ:[[Team:Chiba/protocol#Agarose_gel_electrophoresis]]
 +
 +
#Run the Digested DNA solution
 +
#Cut the agarose target band
 +
#The chip of the gel into 2mL ADB buffer
 +
#Let it in 37 degree 30 min to solve the agarose gel.
 +
#Purify the DNA with Zymo DNA Cleam&Concentrator Kit
 +
 +
READ:[[Team:Chiba/protocol#Zymo_DNA_Cleam&Concentrator_Kit]]
 +
 +
===Dephosphorylation of DNA===
===Dephosphorylation of DNA===
===Phosphorylation of DNA===
===Phosphorylation of DNA===

Revision as of 07:47, 28 August 2009

Home

The Project

1, parallel communication

-LuxR Mutants

2, Series communication

-Las Check

Modeling

Conclusions

Team Parts Reference Notebook Protocols Links Acknowledgements Contact


Protocols

Transformation

Day 1 morning

  • 100 ml SOB medium in 1L or 500 mL flask and sterilize
  • E.coli culture grown in 2 mL of fresh LB medium.

Day 1 night

  • Inoculate preculture (100 μL-1 mL) to sterile SOB medium.Shake culture vigoruoussly at 20-25 ℃ until OD is 0.4-0.6.

Day 2

  • Transfer the culture to ice 10min.
  • Prepare Wash buffer and Competent buffer by adding 3 mL Dilution buffer to 3 mL of Wash buffer(x2) and to 2.5 mL of Competent buffer(2x), respectively.(on ice)
  • Pellet the cells by centrifugarion at 2500rpm for 6 min.
  • Remove supernatant and genlly resupend the cells in 6 mL ice-cold Wash buffer(1x).
  • Pellet the cells by centrifugarion at 2500rpm for 6 min.
  • Completely remove the supernatant and gently resuspend the cells in 6 mL ice-cold Competent buffer(1x).
  • Aliquot (on ice) 100μL of cell suspension into sterile 1.5 mL microtube and store in deep freezer.



DNA Purification

Sigma prep

Zymo DNA Cleam&Concentrator Kit

  • Add 2 volume of DNA binding buffer to each volume of DNA sample, Use voltex to mix.
  • Load mixture silica column and place column into a 2 ml collection tube
  • Centrifuge at full speed for 30 sec. Discard the flow-through.
  • Add 200μL of wash buffer and spin 30 sec.
  • Place siliva volumn into a new 1.5 ml tube. Add water directly to the column matrix and spin to elute the DNA.

Agarose gel electrophoresis

  • Agalose Gel casting
  1. Measure out the appropriate mass of agarose into glass bottle with the appropriate volume of TAE buffer
  2. Microwave until the agarose is fully melted
  3. Pour the agarose solution into the gelbox and let it cool for about 30 minutes, until the gel is solid
  4. Remove comb


  • Running agalose gel
  1. Load 5 μL prepared 1kbp ladder
  2. Mix DNA solution with loading dye(6x) and water
  3. Load it into agalose gel
  4. Run the gel at ~100 volts for 35 mins.
  • Visualizing agarose gels
  1. Remove gel from gel box
  2. Soak the gel in ethidium bromide solution
  3. Let it 30 min.
  4. Place the gel in Trans-Illuminator and turn on UV light after make sure the door closing.
  5. Print the picture.
  6. Remove gel and throw in trash
  7. Wipe down Trans-Illuminator if necessary.


Digestion

PCR

  • Resuspend primer in Nuculease free water to 100 µM
  • PCR mix
  1. DNA template 1µL
  2. Fwd primer 10µL (final con. 10 pM)
  3. Rev primer 10µL
  4. 10x thermo pol buffer 10µL
  5. dNTP mix 10µL
  6. DNA pol. 1µL
  7. dH20 58µL


----------------------------
100µL


  • PCR cycle 
Start: 94 °C for 5 min. (melt)
cycle:          melt:            1 min. 
             anneal :           30 sec. 
cycle end: extension:72 °C for 3.5 min. 
25 cycles 
72 °C for 10 min
store: keep at 6 °C forever


Gel extract

READ:Team:Chiba/protocol#Agarose_gel_electrophoresis

  1. Run the Digested DNA solution
  2. Cut the agarose target band
  3. The chip of the gel into 2mL ADB buffer
  4. Let it in 37 degree 30 min to solve the agarose gel.
  5. Purify the DNA with Zymo DNA Cleam&Concentrator Kit

READ:Team:Chiba/protocol#Zymo_DNA_Cleam&Concentrator_Kit


Dephosphorylation of DNA

Phosphorylation of DNA

Ligation

Time Delay Test

Retrieved from "http://2009.igem.org/Team:Chiba/Notebook/protocol"