Engineering BioBrick vectors from BioBrick parts/Restriction digest

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(New page: ==Materials== *Restriction enzymes ([http://www.neb.com/nebecomm/products/productR0101.asp EcoRI], [http://www.neb.com/nebecomm/products/productR0133.asp SpeI], [http://www.neb.com/nebeco...)
Current revision (11:41, 18 March 2008) (view source)
 
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*Deionized, sterile H<sub>2</sub>O
*Deionized, sterile H<sub>2</sub>O
*0.5-1 &mu;g DNA
*0.5-1 &mu;g DNA
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We performed all restriction digests by mixing 0.5-1 $\mu$g DNA, 1X NEBuffer 2, 100 $\mu$g/ml Bovine Serum Albumin, and 1 $\mu$L each needed restriction enzyme in a 50 $\mu$L total volume.
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*Sterile 0.6mL plastic tubes
==Equipment==
==Equipment==
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==Procedure==
==Procedure==
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Vortex all reagents before use.
#Add appropriate amount of deionized H<sub>2</sub>O to sterile 0.6 mL tube
#Add appropriate amount of deionized H<sub>2</sub>O to sterile 0.6 mL tube
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#Add restriction enzyme buffer.  <br>Vortex buffer before pipetting to ensure that it is well-mixed.
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#Add restriction enzyme buffer.
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#Add BSA.  <br>Vortex BSA before pipetting to ensure that it is well-mixed.
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#Add BSA.
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#Add DNA. <br>Vortex DNA before pipetting to ensure that it is well-mixed.
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#Add DNA.
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#Add each enzyme. <br>Vortex enzyme before pipetting to ensure that it is well-mixed.  <br>Also, the enzyme is in some percentage of glycerol which tends to stick to the sides of your tip.  To ensure you add only 1 &mu;L, just touch your tip to the surface of the liquid when pipetting.
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#Add each enzyme. <br>Also, the enzyme is in some percentage of glycerol which tends to stick to the sides of your tip.  To ensure you add only 1 &mu;L, just touch your tip to the surface of the liquid when pipetting.
#Incubate for 2 hours at 37&deg;C.
#Incubate for 2 hours at 37&deg;C.
#Incubate for 20 mins at 80&deg;C to heat inactivate enzyme.<br> This step is sufficient to inactivate even Pst I.
#Incubate for 20 mins at 80&deg;C to heat inactivate enzyme.<br> This step is sufficient to inactivate even Pst I.

Current revision

Contents

Materials

  • Restriction enzymes (EcoRI, SpeI, XbaI or PstI) from NEB
  • Bovine Serum Albumin (BSA)
  • Deionized, sterile H2O
  • 0.5-1 μg DNA
  • Sterile 0.6mL plastic tubes

Equipment

  • DNA Engine Peltier Thermal Cycler (PTC-200) from MJ Research, Inc. (now Bio-Rad Laboratories, Inc., Hercules, CA).

Digest mix

  • 1X NEB2 buffer
  • 100 μg/mL BSA
  • 1 μL BioBrick enzyme 1
  • 1 μL BioBrick enzyme 2

deionized, sterile H2O to 50 μL

Procedure

Vortex all reagents before use.

  1. Add appropriate amount of deionized H2O to sterile 0.6 mL tube
  2. Add restriction enzyme buffer.
  3. Add BSA.
  4. Add DNA.
  5. Add each enzyme.
    Also, the enzyme is in some percentage of glycerol which tends to stick to the sides of your tip. To ensure you add only 1 μL, just touch your tip to the surface of the liquid when pipetting.
  6. Incubate for 2 hours at 37°C.
  7. Incubate for 20 mins at 80°C to heat inactivate enzyme.
    This step is sufficient to inactivate even Pst I.
  8. Incubate 4°C until you pull the reaction out of the thermal cycler.
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