Griffitts:Restriction digest: Difference between revisions
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<font size="5">Restriction Digest</font> | <font size="5">Restriction Digest</font> | ||
==Materials== | ==Materials== |
Revision as of 10:08, 11 April 2008
Restriction Digest
Materials
- ddH20
- DNA sample on ice
- Appropriate 10X buffer (see below) on ice
- 10X BSA on ice (if necessary; see below)
- Restriction enzyme(s) (see below) on ice
- CIP (keep in freezer until needed)
Recipes
15 μL reaction
- 5 μL DNA
- 1.8 μL appropriate 10X buffer
- 1.5 μL 10X BSA (if needed--see below)
- Restriction enzyme(s) (see specific enzymes below for exact amounts)
- Don't immerse the tip, draw from the surface to avoid excess enzyme
- Add ddH20 to bring the final volume to 15 μL
Note: Use this recipe when verifying plasmid inserts
Note: You can check this reaction after 1 hour
30 μL reaction
- 12 μL DNA
- 3.3 μL appropriate 10X buffer
- 3.0 μL 10X BSA (if needed--see below)
- Restriction enzyme(s) (see specific enzymes below for exact amounts)
- Don't immerse the tip, draw from the surface to avoid excess enzyme
- Add ddH20 to bring the final volume to 30 μL
Note: Use this recipe when your DNA concentrations are sufficient
40 μL reaction
- 20 μL DNA
- 4.3 μL appropriate 10X buffer
- 4.0 μL 10X BSA (if needed--see below)
- Restriction enzyme(s) (see specific enzymes below for exact amounts)
- Don't immerse the tip, draw from the surface to avoid excess enzyme
- Add ddH20 to bring the final volume to 40 μL
Note: Use this recipe when your DNA concentrations are low
Procedure
- Combine ingredients for recipe
- Incubate at 37°C for 2.5 to 8 hours
- If preparing a ligation, add 0.5 μL CIP to the vector 30 minutes prior to ending the reaction
- When the reaction is complete you can store your samples in the -20°C freezer or proceed to in-gel ligation
Enzymes
Enzyme | Amount | Target | Overhang | NEBuffer1 | NEBuffer2 | NEBuffer3 | NEBuffer4 | Other Buffer | T° | BSA |
---|---|---|---|---|---|---|---|---|---|---|
Avr II | c/ctagg | ctag | 100 | 1001 | 50 | 100 | None | 37°C | No | |
Bam HI | 0.8 μL | g/gatcc | gatc | 75 | 100 | 50 | 75 | Bam HI | 37°C | Yes |
Bgl II | a/gatct | gatc | 10 | 75 | 100 | 10 | None | 37°C | No | |
Bst XI | ccannnnn/ntgg | nnnn | 25 | 100 | 100 | 50 | None | 55°C | No | |
Eco RI | g/aattc | aatt | 100 | 100 | 100 | 100 | Eco RI | 37°C | No | |
Eco RV | gat/atc | none | 50 | 75 | 100 | 50 | None | 37°C | Yes | |
Hind III | a/agctt | agct | 50 | 100 | 10 | 50 | None | 37°C | No | |
Kpn I | ggtac/c | gtac | 100 | 75 | 0 | 75 | None | 37°C | Yes | |
Nde I | ca/tatg | ta | 75 | 100 | 75 | 100 | None | 37°C | No | |
Pst I | ctgca/g | tgca | 75 | 75 | 100 | 50 | None | 37°C | Yes | |
Sac I | gagct/c | agct | 100 | 50 | 10 | 100 | None | 37°C | Yes | |
Sal I | g/tcgac | tcga | 0 | 0 | 100 | 0 | None | 37°C | Yes | |
Sma I | ccc/ggg | none | 0 | 0 | 0 | 100 | None | 25°C | No | |
Spe I | a/ctagt | ctag | 75 | 100 | 25 | 75 | None | 37°C | Yes | |
Sph I | gcatg/c | catg | 100 | 100 | 50 | 100 | None | 37°C | No | |
Xba I | 1.5 μL | t/ctaga | ctag | 0 | 100 | 75 | 75 | None | 37°C | Yes |
Xho I | c/tcgag | tcga | 75 | 100 | 100 | 100 | None | 37°C | Yes |
1Boldface indicates the preferred buffer for the enzyme.
Adapted from New England BioLabs 2005-06 Catalog and Technical Reference.
Double Digests
Enzyme | Avr II | Bam HI | Bgl II | Eco RI | Eco RV | Hind III | Kpn I | Nde I | Pst I | Sac I | Sal I | Sma I | Spe I | Sph I | Xba I |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Bam HI | seq1 | - | - | - | - | - | - | - | - | - | - | - | - | - | - |
Bgl II | 3 | Bam HI | - | - | - | - | - | - | - | - | - | - | - | - | - |
Eco RI | Eco RI | Eco RI | Eco RI | - | - | - | - | - | - | - | - | - | - | - | - |
Eco RV | 2 | Bam HI | 3 | Eco RI | - | - | - | - | - | - | - | - | - | - | - |
Hind III | 2 | seq | 2 | Eco RI | 2 | - | - | - | - | - | - | - | - | - | - |
Kpn I | 1 | seq | 2 | 2 | 2 | 2 | - | - | - | - | - | - | - | - | - |
Nde I | 4 | Bam HI | 3 | Eco RI | 2 | 2 | 1 | - | - | - | - | - | - | - | - |
Pst I | 3 | Bam HI | 3 | Eco RI | 3 | 2 | 1 | 3 | - | - | - | - | - | - | - |
Sac I | 1 | seq | 2 | seq | 2 | 2 | 1 | 4 | 1 | - | - | - | - | - | - |
Sal I | seq | Bam HI | 3 | Eco RI | 3 | seq | seq | 3 | 3 | seq | - | - | - | - | - |
Sma I | 4 | seq | seq | seq | 4 | 4 | seq | 4 | 4 | 4 | seq | - | - | - | - |
Spe I | 2 | Bam HI | 2 | Eco RI | 2 | 2 | 1 | 2 | 2 | 1 | seq | 4 | - | - | - |
Sph I | 2 | Bam HI | 2 | Eco RI | 2 | 2 | 1 | 2 | 2 | 1 | seq | 4 | 2 | - | - |
Xba I | 2 | seq | 2 | 2 | 2 | 2 | 2 | 2 | 3 | 4 | 3 | 4 | 2 | 2 | - |
Xho I | 2 | Bam HI | 3 | Eco RI | 3 | 2 | 1 | 4 | 3 | 1 | 3 | 4 | 2 | 2 | 2 |
1 Sequential digest recommended.
Note that a double-digest of Bam HI and Xba I is not recommended. However, by following the procedure above you won't have any problems.
Adapted from New England BioLabs 2005-06 Catalog and Technical Reference.