Griffitts:Restriction digest: Difference between revisions
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* 5 μL DNA | * 5 μL DNA | ||
* 1.5 μL appropriate 10X buffer | * 1.5 μL appropriate 10X buffer | ||
* 1.5 μL 10X BSA (if needed | * 1.5 μL 10X BSA (if needed—see below) | ||
* Restriction enzyme(s) (see specific enzymes below for exact amounts) | * Restriction enzyme(s) (see specific enzymes below for exact amounts) | ||
** Don't immerse the tip, draw from the surface to avoid excess enzyme | ** Don't immerse the tip, draw from the surface to avoid excess enzyme | ||
Line 24: | Line 24: | ||
===30 μL reaction=== | ===30 μL reaction=== | ||
* 12 μL DNA | * 12 μL DNA | ||
* 3. | * 3.0 μL appropriate 10X buffer | ||
* 3.0 μL 10X BSA (if needed | * 3.0 μL 10X BSA (if needed—see below) | ||
* Restriction enzyme(s) (see specific enzymes below for exact amounts) | * Restriction enzyme(s) (see specific enzymes below for exact amounts) | ||
** Don't immerse the tip, draw from the surface to avoid excess enzyme | ** Don't immerse the tip, draw from the surface to avoid excess enzyme | ||
* Add ddH<sub>2</sub>0 to bring the final volume to 30 μL | * Add ddH<sub>2</sub>0 to bring the final volume to 30 μL | ||
Note: Use this recipe when your DNA concentrations are sufficient | Note: Use this recipe when your DNA concentrations are sufficient<br> | ||
Note: For a ''Bam''HI–''Xba''I double-digest, use 3.3 μL of 10X buffer | |||
===40 μL reaction=== | ===40 μL reaction=== | ||
* 20 μL DNA | * 20 μL DNA | ||
* 4. | * 4.0 μL appropriate 10X buffer | ||
* 4.0 μL 10X BSA (if needed | * 4.0 μL 10X BSA (if needed—see below) | ||
* Restriction enzyme(s) (see specific enzymes below for exact amounts) | * Restriction enzyme(s) (see specific enzymes below for exact amounts) | ||
** Don't immerse the tip, draw from the surface to avoid excess enzyme | ** Don't immerse the tip, draw from the surface to avoid excess enzyme | ||
* Add ddH<sub>2</sub>0 to bring the final volume to 40 μL | * Add ddH<sub>2</sub>0 to bring the final volume to 40 μL | ||
Note: Use this recipe when your DNA concentrations are low | Note: Use this recipe when your DNA concentrations are low<br> | ||
Note: For a ''Bam''HI–''Xba''I double-digest, use 4.3 μL of 10X buffer | |||
==Procedure== | ==Procedure== | ||
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| '''100''' | | '''100''' | ||
| 75 | | 75 | ||
| | | '''100''' | ||
| None | | None | ||
| 37°C | | 37°C | ||
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|- | |- | ||
| '''''Bam'' HI''' | | '''''Bam'' HI''' | ||
| seq<sup>1</sup> | | seq<sup><font color=red>1</font></sup> | ||
| - | | - | ||
| - | | - | ||
Line 515: | Line 517: | ||
| '''''Xba'' I''' | | '''''Xba'' I''' | ||
| 2 | | 2 | ||
| seq | | seq<sup><font color=red>2</font></sup> | ||
| 2 | | 2 | ||
| 2 | | 2 | ||
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|- | |- | ||
|} | |} | ||
<sup>1</sup> Sequential digest recommended.<br> | <sup><font color=red>1</font></sup>Sequential digest recommended.<br> | ||
<sup><font color=red>2</font></sup>We actually prefer to do the double-digest and use Buffer 2 for the ''Bam''HI–''Xba''I double-digest.<br> | |||
Note that a double-digest of ''Bam'' HI and ''Xba'' I is not recommended. However, by following [[Griffitts:Restriction digest#30 μL reaction|the procedure above]] you won't have any problems. | Note that a double-digest of ''Bam'' HI and ''Xba'' I is not recommended. However, by following [[Griffitts:Restriction digest#30 μL reaction|the procedure above]] you won't have any problems. | ||
<br><br> | <br><br> | ||
'' | Adapted from ''New England BioLabs 2005-06 Catalog and Technical Reference.'' |
Latest revision as of 11:38, 20 March 2012
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
- 1.5 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
- 3.0 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
Note: For a BamHI–XbaI double-digest, use 3.3 μL of 10X buffer
40 μL reaction
- 20 μL DNA
- 4.0 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
Note: For a BamHI–XbaI double-digest, use 4.3 μL of 10X buffer
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 | 100 | 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 | seq2 | 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 |
1Sequential digest recommended.
2We actually prefer to do the double-digest and use Buffer 2 for the BamHI–XbaI double-digest.
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.