Team:Brown/Protocols/Restriction Digest

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Revision as of 05:24, 25 October 2010

Restriction Digest protocol

Brown iGEM 2010, modified from Knight Lab protocol.

Materials

Restriction enzymes (we use NEB)
NEB buffer 2/3 (use buffer finder on NEB website. Buffer 2 is standard for iGEM enzymes)
BSA
Deionized, sterile H2O

Digest mix

The reaction below is a 50ul reaction, 100ul reactions are also common if you want your DNA to be dilute.

5ul NEB Buffer 2
Xul DNA (usually ~500ng)
0.5ul 100x BSA
1ul Restriction Enzyme (regardless of the reaction, 1ul is used because this generally represents a 10-25 fold excess. It is also sticky due to its glycerol content, so pipetting can be difficult)
1ul Restriction Enzyme 2 (for double digests)
(42.5-X)ul dH2O

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.
1. 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.
2. 20 mins at 80°C to heat inactivate enzyme.
  - This step is sufficient to inactivate even Pst I.
3. 4°C forever (or until you pull the reaction out of the thermal cycler).
Generally, use some method of DNA purification to eliminate enzymes and salt from the reaction.

@@ Line 14: / Line 14: @@
 The reaction below is a 50ul reaction, 100ul reactions are also common if you want your DNA to be dilute.
 *5ul NEB Buffer 2
-*_ul DNA (usually ~500ng)
+*Xul DNA (usually ~500ng)
 *0.5ul 100x BSA
-*
+*1ul Restriction Enzyme (regardless of the reaction, 1ul is used because this generally represents a 10-25 fold excess. It is also sticky due to its glycerol content, so pipetting can be difficult)
-*
+*1ul Restriction Enzyme 2 (for double digests)
-*
+*(42.5-X)ul dH2O
+#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).
+#Generally, use some method of DNA purification to eliminate enzymes and salt from the reaction.