Team:British Columbia/Protocols/Restriction Digests

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{{Template:Team:British Columbia Header}}
{{Template:Team:British Columbia Header}}
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=Restriction Digest Protocol=
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=Restriction Digests=
1. Prepare reaction mix, following the basic ratios below. Each reaction requires at least 4 uL of master mix.
1. Prepare reaction mix, following the basic ratios below. Each reaction requires at least 4 uL of master mix.
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{| class="wikitable"
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|+ Master Mix
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! Ratio !! Name of Reagent !! Quantity/reaction (uL)
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|-
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| 5x || NEB Buffer 2 || 1
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|-
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| 0.5x || BSA || 0.1
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|-
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| 0.5x || Dpn1 || 0.1
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|-
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| 0.5x || Eco-RI (HF) || 0.1
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|-
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| 0.5x || Pst1 || 0.1
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|-
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| 18x || dH<sub>2</sub>O || 3.6
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|-
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|TOTAL || || 5 uL
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|}
Notes:
Notes:

Revision as of 00:48, 26 June 2012

British Columbia - 2012.igem.org

Restriction Digests

1. Prepare reaction mix, following the basic ratios below. Each reaction requires at least 4 uL of master mix.

Master Mix
Ratio Name of Reagent Quantity/reaction (uL)
5x NEB Buffer 2 1
0.5x BSA 0.1
0.5x Dpn1 0.1
0.5x Eco-RI (HF) 0.1
0.5x Pst1 0.1
18x dH2O 3.6
TOTAL 5 uL

Notes:

  • do not add DPN1 when working with plasmid DNA - it will cut all methylated DNA.
  • keep the restriction enzymes as close to -20°C as possible.

2. Add 4 uL of master mix and 4 uL of undigested template DNA (plasmid or PCR product) to a 200 uL PCR tube.

3. Heat the mixtures at 37°C for 30 minutes for optimal enzyme activity, then 80°C for 20 minutes to inactivate the enzymes.

  • easiest to use a thermocycler
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