Engineering BioBrick vectors from BioBrick parts/Restriction digest: Difference between revisions
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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...) |
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*Deionized, sterile H<sub>2</sub>O | *Deionized, sterile H<sub>2</sub>O | ||
*0.5-1 μg DNA | *0.5-1 μg DNA | ||
*Sterile 0.6mL plastic tubes | |||
==Equipment== | ==Equipment== | ||
Line 20: | Line 20: | ||
==Procedure== | ==Procedure== | ||
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 | ||
#Add restriction enzyme buffer | #Add restriction enzyme buffer. | ||
#Add BSA | #Add BSA. | ||
#Add DNA | #Add DNA. | ||
#Add each enzyme. | #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 μL, just touch your tip to the surface of the liquid when pipetting. | ||
#Incubate for 2 hours at 37°C. | #Incubate for 2 hours at 37°C. | ||
#Incubate for 20 mins at 80°C to heat inactivate enzyme.<br> This step is sufficient to inactivate even Pst I. | #Incubate for 20 mins at 80°C to heat inactivate enzyme.<br> This step is sufficient to inactivate even Pst I. |
Latest revision as of 08:41, 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.
- Add appropriate amount of deionized H2O to sterile 0.6 mL tube
- Add restriction enzyme buffer.
- Add BSA.
- Add DNA.
- 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. - Incubate for 2 hours at 37°C.
- Incubate for 20 mins at 80°C to heat inactivate enzyme.
This step is sufficient to inactivate even Pst I. - Incubate 4°C until you pull the reaction out of the thermal cycler.