Knight:Restriction Digest: Difference between revisions
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*Restriction enzymes ([http://www.neb.com/nebecomm/products/productR0101.asp EcoR I], [http://www.neb.com/nebecomm/products/productR0133.asp Spe I], [http://www.neb.com/nebecomm/products/productR0145.asp Xba I] or [http://www.neb.com/nebecomm/products/productR0140.asp Pst I]) from [http://www.neb.com/ NEB] | *Restriction enzymes ([http://www.neb.com/nebecomm/products/productR0101.asp EcoR I], [http://www.neb.com/nebecomm/products/productR0133.asp Spe I], [http://www.neb.com/nebecomm/products/productR0145.asp Xba I] or [http://www.neb.com/nebecomm/products/productR0140.asp Pst I]) from [http://www.neb.com/ NEB] | ||
* | *NEB2 buffer | ||
*BSA | *BSA | ||
*Deionized, sterile H<sub>2</sub>O | *Deionized, sterile H<sub>2</sub>O | ||
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''Example - 50 μL reaction. 100 μL reactions are also common especially if your DNA to be cut is dilute.'' | ''Example - 50 μL reaction. 100 μL reactions are also common especially if your DNA to be cut is dilute.'' | ||
*5 μL | *5 μL NEB2 buffer (for all digests with BioBricks enzymes, we use NEB2 buffer. It keeps things simple and seems to work). | ||
*X μL DNA (usually ~ | *X μL DNA (usually ~500 ng depending on downstream uses). | ||
*0.5 μL 100X BSA (added to all digests because BSA never hurts a restriction digest) | *0.5 μL 100X BSA (added to all digests because BSA never hurts a restriction digest) | ||
*1 μL BioBricks enzyme 1 (regardless of the volume of the reaction, 1 μL enzyme is used because generally this represents a 10-25 fold excess of enzyme and is therefore sufficient for most digests. Also, it can be difficult to accurately pipet less than 1 μL of enzyme since it is sticky due to the glycerol content.) | *1 μL BioBricks enzyme 1 (regardless of the volume of the reaction, 1 μL enzyme is used because generally this represents a 10-25 fold excess of enzyme and is therefore sufficient for most digests. Also, it can be difficult to accurately pipet less than 1 μL of enzyme since it is sticky due to the glycerol content.) | ||
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#Add 1 μL of 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 μL, just touch your tip to the surface of the liquid when pipetting. | #Add 1 μL of 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 μL, just touch your tip to the surface of the liquid when pipetting. | ||
#Place in thermal cycler ([http://www.mjr.com/ MJ Research], PT-200) and run digest protocol. | #Place in thermal cycler ([http://www.mjr.com/ MJ Research], PT-200) and run digest protocol. | ||
##4-6 hour incubation at 37°C <br> Use a longer incubation time if you have time or are worried about the efficiency of cutting. | ##4-6 hour incubation at 37°C <br> Use a longer incubation time if you have time or are worried about the efficiency of cutting. I think this time can be shortened to 2 hrs while still cutting to completion. | ||
##20 mins at 80°C to heat inactivate enzyme.<br> This step is sufficient to inactivate even Pst I. | ##20 mins at 80°C to heat inactivate enzyme.<br> This step is sufficient to inactivate even Pst I. | ||
##4°C forever (or until you pull the reaction out of the thermal cycler). | ##4°C forever (or until you pull the reaction out of the thermal cycler). | ||
#Generally, use some method of [[Purification of DNA | DNA purification]] to eliminate enzymes and salt from the reaction. | #Generally, use some method of [[Purification of DNA | DNA purification]] to eliminate enzymes and salt from the reaction. | ||
[[Category:Protocol]] [[Category:DNA]] [[Category:In vitro]] |
Latest revision as of 08:22, 18 March 2008
Materials
Digest Mix
Example - 50 μL reaction. 100 μL reactions are also common especially if your DNA to be cut is dilute.
- 5 μL NEB2 buffer (for all digests with BioBricks enzymes, we use NEB2 buffer. It keeps things simple and seems to work).
- X μL DNA (usually ~500 ng depending on downstream uses).
- 0.5 μL 100X BSA (added to all digests because BSA never hurts a restriction digest)
- 1 μL BioBricks enzyme 1 (regardless of the volume of the reaction, 1 μL enzyme is used because generally this represents a 10-25 fold excess of enzyme and is therefore sufficient for most digests. Also, it can be difficult to accurately pipet less than 1 μL of enzyme since it is sticky due to the glycerol content.)
- 1 μL BioBricks enzyme 2
- (42.5 - X) μL deionized, sterile H2O
Procedure
- Add appropriate amount of deionized H2O to sterile 0.6 mL tube
- Add restriction enzyme buffer to the tube.
Vortex buffer before pipetting to ensure that it is well-mixed. - Add BSA to the tube.
Vortex BSA before pipetting to ensure that it is well-mixed. - Add appropriate amount of DNA to be cut to the tube.
Vortex DNA before pipetting to ensure that it is well-mixed. - Add 1 μL of each enzyme.
Vortex enzyme before pipetting to ensure that it is well-mixed.
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. - Place in thermal cycler (MJ Research, PT-200) and run digest protocol.
- 4-6 hour incubation at 37°C
Use a longer incubation time if you have time or are worried about the efficiency of cutting. I think this time can be shortened to 2 hrs while still cutting to completion. - 20 mins at 80°C to heat inactivate enzyme.
This step is sufficient to inactivate even Pst I. - 4°C forever (or until you pull the reaction out of the thermal cycler).
- 4-6 hour incubation at 37°C
- Generally, use some method of DNA purification to eliminate enzymes and salt from the reaction.