Biomod/2013/Harvard/protocols: Difference between revisions

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=== DNA Clean and Concentrate ===  
=== DNA Clean & Concentrator ===  


This protocol is used to clean and concentrate PRC products after DPN1 digestion. We use the ZYMO DNA Clean & Concentrator - 5 Kit for this protocol.  
ZYMO DNA Clean & Concentrator - 5 Kit was used to purify DNA from PCR.  


# Add 2-7 Volumes of DNA Binding Buffer (from kit) to each volume of DNA sample.
Protocol:
# Add 2-7 Volumes of DNA Binding Buffer to each volume of DNA sample.
## For plasmid or genomic DNA 2kb, add 2 volumes.  
## For plasmid or genomic DNA 2kb, add 2 volumes.  
## For PCR or short DNA fragments, add 5 volumes.  
## For PCR or short DNA fragments, add 5 volumes.  
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# Load the mixture into a Zymo-Spin Column in a Collection Tube.  
# Load the mixture into a Zymo-Spin Column in a Collection Tube.  
# Centrifuge at full speed (≥10,000 x g) for 30 seconds. Discard flow through.  
# Centrifuge at full speed (≥10,000 x g) for 30 seconds. Discard flow through.  
# Add 200 µl of DNA Wash Buffer (from kit) to the column and centrifuge for 30 seconds. Repeat the wash step, this time centrifuging for 1 minute.  
# Add 200 µl of DNA Wash Buffer to the column and centrifuge for 30 seconds.
# Place the Zymo-Spin Column into a new 1.5 ml tube. Add ≥6 µl of DNA Elution Buffer (from kit) or water directly to the column matrix and spin to elute the DNA.
# Repeat previous step, this time centrifuging for 1 minute.  
# Place the Zymo-Spin Column into a new 1.5 ml tube. Add ≥6 µl of DNA Elution Buffer or water directly to the column matrix and spin (30 seconds) to elute the DNA.


=== Bacterial Cell Culture Solutions ===  
=== Bacterial Cell Culture Solutions ===  

Revision as of 13:10, 17 June 2013

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Protocols

DNA Clean & Concentrator

ZYMO DNA Clean & Concentrator - 5 Kit was used to purify DNA from PCR.

Protocol:

  1. Add 2-7 Volumes of DNA Binding Buffer to each volume of DNA sample.
    1. For plasmid or genomic DNA 2kb, add 2 volumes.
    2. For PCR or short DNA fragments, add 5 volumes.
    3. For ssDNA purification, add 7 volumes
  2. Load the mixture into a Zymo-Spin Column in a Collection Tube.
  3. Centrifuge at full speed (≥10,000 x g) for 30 seconds. Discard flow through.
  4. Add 200 µl of DNA Wash Buffer to the column and centrifuge for 30 seconds.
  5. Repeat previous step, this time centrifuging for 1 minute.
  6. Place the Zymo-Spin Column into a new 1.5 ml tube. Add ≥6 µl of DNA Elution Buffer or water directly to the column matrix and spin (30 seconds) to elute the DNA.

Bacterial Cell Culture Solutions

BLACaM Assays

Chemically Competent Cells Transformation

  1. Thaw chemically competent cells on ice.
  2. Transfer 50 µl of competent cells to a 1.5 ml microcentrifuge tube (if necessary).
  3. Add 2 µl of assembled product to NEB competent cells.
  4. Mix gently by pipetting up and down or flicking the tube 4-5 times. Do not vortex. Place the mixture on ice for 30 minutes. Do not mix.
  5. Heat shock at 42°C for 30 seconds. Do not mix.
  6. Transfer tubes on ice for 2 minutes.
  7. Add 950 µl of room temperature SOC media to tubes.
  8. Place the tube at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
  9. Warm selection plates to 37°C.
  10. Spread 100 µl of the cells onto the plates with appropriate antibiotics. Use amp plates for positive control sample.
  11. Incubate plates overnight at 37°C.

miniprep

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