Difference between revisions of "Engineering BioBrick vectors from BioBrick parts/Restriction digest"

From OpenWetWare
Jump to: navigation, search
(Procedure)
 
(One intermediate revision by the same user not shown)
Line 5: Line 5:
 
*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
 +
*Sterile 0.6mL plastic tubes
  
 
==Equipment==
 
==Equipment==
Line 19: Line 20:
  
 
==Procedure==
 
==Procedure==
 +
 
Vortex all reagents before use.
 
Vortex all reagents before use.
  

Latest revision as of 07:41, 18 March 2008

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.