Team:TU Delft/protocols/restriction enzyme digestion

From 2010.igem.org

(Difference between revisions)
 
(5 intermediate revisions not shown)
Line 1: Line 1:
-
==Restriction enzyme digestion==
+
=Restriction enzyme digestion=
-
Digestions (cutting plasmid DNA) were performed at the appropriate temperature with the appropriate buffer in the appropriate concentration, according to the supplier.
 
-
Restriction enzymes we use are: EcoRI, XbaI, SpeI, PstI
 
 +
''Materials:''
 +
 +
- plasmid DNA or PCR product
 +
 +
- restriction enzymes (Roche and BioLabs)
 +
 +
- buffer (10x)
 +
 +
- H2O
 +
 +
- water bath at 37 °C
 +
 +
- heat block or water bath at 80 °C
 +
 +
 +
''Protocol:''
 +
 +
Digestions (cutting plasmid DNA) were performed at the appropriate temperature with the appropriate buffer in the appropriate concentration, according to the supplier.
Reaction for one sample:
Reaction for one sample:
Line 11: Line 27:
|-
|-
|Buffer (10x)
|Buffer (10x)
-
| 2,0 μL (for 1×)
+
|x μL (for 1×)
|-
|-
|Restriction enzymes
|Restriction enzymes
-
|x μL (5 units/μg DNA = 0.5 µL)
+
|x μL (10 units/μg DNA = 1 µL)
|-
|-
|H2O
|H2O
Line 20: Line 36:
|-
|-
|
|
-
|20 μL
+
|20-25 μL
|}
|}
-
Incubate for (at least) one hour at 37 °C.  Inactivate the restriction endonucleases by heat, incubation at 80 °C for 10 minutes and centrifuge shortly.
+
Incubate for (at least) one hour at 37 °C.  Inactivate the restriction endonucleases by heat, incubation at 80°C for 10 minutes and centrifuge shortly.
 +
 
 +
 
 +
''Used Buffers:''
 +
 
 +
Buffer H (Roche): 50 mM Tris-HCl, 1 M NaCl, 100 mM MgCl2, 10 mM DTE, pH 7.5 at 37 °C
 +
 
 +
Buffer M (Roche): 100 mM Tris-HCl, 500 mM NaCI, 100 mM MgCl2, 10 mM DTE, pH 7.5 at 37 °C
 +
 
 +
Buffer 1 (BioLabs): 10 mM Bis-Tris-Propane-HCl, 10 mM MgCl2, 1 mM DTE,pH 7.0 at 25°C
 +
 
 +
Buffer 2 (BioLabs): 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM DTE, pH 7.9 at 25°C
 +
Buffer 3 (BioLabs): 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 1 mM DTE, pH 7.9 at 25°C
-
Used Buffers:
+
Buffer 4 (BioLabs): 50 mM CH3CO2K, 20 mM TAE, 10 mM Mg(CH3COO)2, 1 mM DTE, pH 7.9 at 25°C
-
Buffer H (Roche): 0.5 M Tris-HCl, 1 M NaCl, 100 mM MgCl2, 10 mM DTE, pH 7.5
+
Note: some of the restriction enzymes of New England BioLabs required the addition of 100 µg/mL BSA

Latest revision as of 18:53, 12 September 2010

Restriction enzyme digestion

Materials:

- plasmid DNA or PCR product

- restriction enzymes (Roche and BioLabs)

- buffer (10x)

- H2O

- water bath at 37 °C

- heat block or water bath at 80 °C


Protocol:

Digestions (cutting plasmid DNA) were performed at the appropriate temperature with the appropriate buffer in the appropriate concentration, according to the supplier.

Reaction for one sample:

DNA x μL (up to 1,0 μg)
Buffer (10x) x μL (for 1×)
Restriction enzymes x μL (10 units/μg DNA = 1 µL)
H2O x μL
20-25 μL


Incubate for (at least) one hour at 37 °C. Inactivate the restriction endonucleases by heat, incubation at 80°C for 10 minutes and centrifuge shortly.


Used Buffers:

Buffer H (Roche): 50 mM Tris-HCl, 1 M NaCl, 100 mM MgCl2, 10 mM DTE, pH 7.5 at 37 °C

Buffer M (Roche): 100 mM Tris-HCl, 500 mM NaCI, 100 mM MgCl2, 10 mM DTE, pH 7.5 at 37 °C

Buffer 1 (BioLabs): 10 mM Bis-Tris-Propane-HCl, 10 mM MgCl2, 1 mM DTE,pH 7.0 at 25°C

Buffer 2 (BioLabs): 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM DTE, pH 7.9 at 25°C

Buffer 3 (BioLabs): 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 1 mM DTE, pH 7.9 at 25°C

Buffer 4 (BioLabs): 50 mM CH3CO2K, 20 mM TAE, 10 mM Mg(CH3COO)2, 1 mM DTE, pH 7.9 at 25°C

Note: some of the restriction enzymes of New England BioLabs required the addition of 100 µg/mL BSA