Team:TU Delft/protocols/restriction enzyme digestion

From 2010.igem.org

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==Restriction enzyme digestion==
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=Restriction enzyme digestion=
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Digestions (cutting plasmid DNA) were performed at the appropriate temperature with the appropriate buffer in the appropriate concentration, according to the supplier.
 
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Restriction enzymes we use are: EcoRI, XbaI, SpeI, PstI
 
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''Materials:''
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- plasmid DNA or PCR product
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- restriction enzymes (Roche and BioLabs)
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- buffer (10x)
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- H2O
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- water bath at 37 °C
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- heat block or water bath at 80 °C
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''Protocol:''
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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:
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|Buffer (10x)
|Buffer (10x)
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| 2,0 μL (for 1×)
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|x μL (for 1×)
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|Restriction enzymes
|Restriction enzymes
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|20 μL
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|20-25 μL
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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.
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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:''
''Used Buffers:''
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Buffer H (Roche): 0.5 M Tris-HCl, 1 M NaCl, 100 mM MgCl2, 10 mM DTE, pH 7.5
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Buffer H (Roche): 50 mM Tris-HCl, 1 M NaCl, 100 mM MgCl2, 10 mM DTE, pH 7.5 at 37 °C
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Buffer M (Roche): 100 mM Tris-HCl, 500 mM NaCI, 100 mM MgCl2, 10 mM DTE, pH 7.5 at 37 °C
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Buffer 1 (BioLabs): 10 mM Bis-Tris-Propane-HCl, 10 mM MgCl2, 1 mM DTE,pH 7.0 at 25°C
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Buffer 2 (BioLabs): 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM DTE, pH 7.9 at 25°C
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1x Buffer 2 (BioLabs): 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM Dithiothreitol, pH 7.9
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Buffer 3 (BioLabs): 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 1 mM DTE, pH 7.9 at 25°C
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1x Buffer 3 (BioLabs): 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 1 mM Dithiothreitol, pH 7.9  
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Buffer 4 (BioLabs): 50 mM CH3CO2K, 20 mM TAE, 10 mM Mg(CH3COO)2, 1 mM DTE, pH 7.9 at 25°C
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Note that the enzymes of BioLabs sometimes 0.01 mg/mL BSA require
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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

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