Knight:Restriction Digest: Difference between revisions
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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. |
Revision as of 13:19, 9 October 2006
Materials
- Restriction enzymes (EcoR I, Spe I, Xba I or Pst I) from NEB
- EcoR I buffer
- BSA
- Deionized, sterile H2O
Digest Mix
Example - 50 μL reaction. 100 μL reactions are also common especially if your DNA to be cut is dilute.
- 5 μL EcoR I buffer (for all digests with BioBricks enzymes, we use EcoR I buffer. It keeps things simple and seems to work).
- X μL DNA (usually ~1 μg 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.