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Team:British Columbia/Protocols/Restriction Digests
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<h1>Restriction Digests</h1> | <h1>Restriction Digests</h1> | ||
| - | 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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| - | Notes: | + | :Notes: |
| - | *do not add Dpn1 when working with plasmid DNA - it will cut all methylated DNA. | + | ::*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 for as long as possible. | + | ::*keep the restriction enzymes as close to -20°C as possible for as long 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. | + | :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. | |
| - | 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 |
| - | *easiest to use a thermocycler | + | :4. Follow up with ligation or store products at -20°C. |
| - | + | ||
| - | 4. Follow up with ligation or store products at -20°C. | + | |
Latest revision as of 01:36, 4 October 2012
Restriction Digests
- 1. Prepare reaction mix, following the basic ratios below. Each reaction requires at least 4 uL of 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 for as long 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
- 4. Follow up with ligation or store products at -20°C.
Ligations
(as provided by Gingko Bioworks)
- Thaw 10X T4 DNA Ligase Reaction Buffer (agitate to force precipitate into solution).
- Add 11 uL of H2O to a 200 ul PCR tube.
- Add 3 uL of the digested insert and 1 uL of the digested vector.
- Add 2 uL of 10X T4 DNA Ligase Reaction Buffer.
- Add 1 uL of T4 DNA Ligase enzyme (thawed).
- Mix the reagents by flicking the tube. Spin briefly in the microcentrifuge to collect the liquid in the bottom of the tube. The total volume in the tube should be 20 uL.
- Let the mix sit at room temperature for 30 minutes, followed by heat inactivation at 80°C for 20 minutes.
- Use the product for transformation.
