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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''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:''
Digestions (cutting plasmid DNA) were performed at the appropriate temperature with the appropriate buffer in the appropriate concentration, according to the supplier.
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
 
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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|x μL (5 units/μg DNA = 0.5 µL)
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|x μL (10 units/μg DNA = 1 µL)
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|-
|H2O
|H2O
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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.
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 +
 
 +
''Used Buffers:''
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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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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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Used Buffers:
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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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Buffer H (Roche): 0.5 M Tris-HCl, 1 M NaCl, 100 mM MgCl2, 10 mM DTE, pH 7.5
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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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