Griffitts:Restriction digest: Difference between revisions
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{{Griffitts}} | <div class="noprint"> | ||
{{Template:Griffitts}} | |||
</div> | |||
<font size="5">Restriction Digest</font> | <font size="5">Restriction Digest</font> | ||
==Materials== | ==Materials== | ||
* ddH<sub>2</sub>0 | * ddH<sub>2</sub>0 | ||
* DNA sample on ice | * DNA sample on ice | ||
* Appropriate 10X buffer on ice | * Appropriate 10X buffer (see below) on ice | ||
* 10X BSA on ice | * 10X BSA on ice (if necessary; see below) | ||
* Restriction enzyme(s) (see below) on ice | * Restriction enzyme(s) (see below) on ice | ||
* CIP (keep in freezer until needed) | * CIP (keep in freezer until needed) | ||
== | |||
* 12-20 μL DNA | ==Recipes== | ||
* 4. | ===15 μL reaction=== | ||
* 4.0 μL 10X BSA | * 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 ddH<sub>2</sub>0 to bring the final volume to 15 μL | |||
Note: Use this recipe when verifying plasmid inserts<br> | |||
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 ddH<sub>2</sub>0 to bring the final volume to 30 μL | |||
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=== | |||
* 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) | * 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<br> | |||
Note: For a ''Bam''HI–''Xba''I double-digest, use 4.3 μL of 10X buffer | |||
==Procedure== | ==Procedure== | ||
* Combine ingredients for recipe | * Combine ingredients for recipe | ||
* Incubate at 37°C for 2.5 to 8 hours | * 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 | * 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 | * When the reaction is complete you can store your samples in the -20°C freezer or proceed to [[Griffitts:In-gel ligation|in-gel ligation]] | ||
==Enzymes== | ==Enzymes== | ||
{| border="1" cellpadding="5" cellspacing="0" class="wikitable sortable" | {| border="1" cellpadding="5" cellspacing="0" class="wikitable sortable" | ||
Line 25: | Line 53: | ||
|- | |- | ||
! width="30" style="background:#efefef;" | Enzyme | ! width="30" style="background:#efefef;" | Enzyme | ||
! width=" | ! width="30" style="background:#efefef;" | Amount | ||
! width="90" style="background:#efefef;" | Target | ! width="90" style="background:#efefef;" | Target | ||
! width=" | ! width="30" style="background:#efefef;" | Overhang | ||
! width="60" style="background:#efefef;" | NEBuffer1 | ! width="60" style="background:#efefef;" | NEBuffer1 | ||
! width="60" style="background:#efefef;" | NEBuffer2 | ! width="60" style="background:#efefef;" | NEBuffer2 | ||
Line 33: | Line 61: | ||
! width="60" style="background:#efefef;" | NEBuffer4 | ! width="60" style="background:#efefef;" | NEBuffer4 | ||
! width="60" style="background:#efefef;" | Other Buffer | ! width="60" style="background:#efefef;" | Other Buffer | ||
! width=" | ! width="30" style="background:#efefef;" | T° | ||
! width=" | ! width="30" style="background:#efefef;" | BSA | ||
|- | |- | ||
| ''Avr''II | | ''Avr'' II | ||
| | | | ||
| c/ctagg | | c/ctagg | ||
| ctag | | ctag | ||
| 100 | | 100 | ||
| '''100''' | | '''100'''<sup>1</sup> | ||
| 50 | | 50 | ||
| 100 | | 100 | ||
Line 48: | Line 76: | ||
| No | | No | ||
|- | |- | ||
| ''Bam''HI | | ''Bam'' HI | ||
| | | 0.8 μL | ||
| g/gatcc | | g/gatcc | ||
| gatc | | gatc | ||
Line 56: | Line 84: | ||
| 50 | | 50 | ||
| 75 | | 75 | ||
| ''Bam''HI | | '''''Bam'' HI''' | ||
| 37°C | | 37°C | ||
| Yes | | Yes | ||
|- | |- | ||
| ''Bgl''II | | ''Bgl'' II | ||
| | | | ||
| a/gatct | | a/gatct | ||
Line 72: | Line 100: | ||
| No | | No | ||
|- | |- | ||
| ''Bst''XI | | ''Bst'' XI | ||
| | | | ||
| ccannnnn/ntgg | | ccannnnn/ntgg | ||
Line 84: | Line 112: | ||
| No | | No | ||
|- | |- | ||
| ''Eco''RI | | ''Eco'' RI | ||
| | | | ||
| g/aattc | | g/aattc | ||
Line 92: | Line 120: | ||
| 100 | | 100 | ||
| 100 | | 100 | ||
| ''Eco''RI | | '''''Eco'' RI''' | ||
| 37°C | | 37°C | ||
| No | | No | ||
|- | |- | ||
| ''Eco''RV | | ''Eco'' RV | ||
| | | | ||
| gat/atc | | gat/atc | ||
Line 108: | Line 136: | ||
| Yes | | Yes | ||
|- | |- | ||
| ''Hind''III | | ''Hind'' III | ||
| | | | ||
| a/agctt | | a/agctt | ||
Line 120: | Line 148: | ||
| No | | No | ||
|- | |- | ||
| ''Kpn''I | | ''Kpn'' I | ||
| | | | ||
| ggtac/c | | ggtac/c | ||
Line 132: | Line 160: | ||
| Yes | | Yes | ||
|- | |- | ||
| ''Nde''I | | ''Nde'' I | ||
| | | | ||
| ca/tatg | | ca/tatg | ||
Line 144: | Line 172: | ||
| No | | No | ||
|- | |- | ||
| ''Pst''I | | ''Pst'' I | ||
| | | | ||
| ctgca/g | | ctgca/g | ||
Line 156: | Line 184: | ||
| Yes | | Yes | ||
|- | |- | ||
| ''Sac''I | | ''Sac'' I | ||
| | | | ||
| gagct/c | | gagct/c | ||
Line 168: | Line 196: | ||
| Yes | | Yes | ||
|- | |- | ||
| ''Sal''I | | ''Sal'' I | ||
| | | | ||
| g/tcgac | | g/tcgac | ||
Line 180: | Line 208: | ||
| Yes | | Yes | ||
|- | |- | ||
| ''Sma''I | | ''Sma'' I | ||
| | | | ||
| ccc/ggg | | ccc/ggg | ||
Line 192: | Line 220: | ||
| No | | No | ||
|- | |- | ||
| ''Spe''I | | ''Spe'' I | ||
| | | | ||
| a/ctagt | | a/ctagt | ||
Line 204: | Line 232: | ||
| Yes | | Yes | ||
|- | |- | ||
| ''Sph''I | | ''Sph'' I | ||
| | | | ||
| gcatg/c | | gcatg/c | ||
Line 216: | Line 244: | ||
| No | | No | ||
|- | |- | ||
| ''Xba''I | | ''Xba'' I | ||
| 1.5 μL | | 1.5 μL | ||
| t/ctaga | | t/ctaga | ||
Line 223: | Line 251: | ||
| '''100''' | | '''100''' | ||
| 75 | | 75 | ||
| | | '''100''' | ||
| None | | None | ||
| 37°C | | 37°C | ||
| Yes | | Yes | ||
|- | |- | ||
| ''Xho''I | | ''Xho'' I | ||
| | | | ||
| c/tcgag | | c/tcgag | ||
Line 241: | Line 269: | ||
|- | |- | ||
|} | |} | ||
<sup>1</sup>'''Boldface''' indicates the preferred buffer for the enzyme. | |||
<br><br> | |||
''Adapted from New England BioLabs 2005-06 Catalog and Technical Reference.'' | |||
==Double Digests== | |||
{| border="1" cellpadding="5" cellspacing="0" | |||
|+'''Suggested Buffers''' | |||
|- | |||
! width="30" style="background:#efefef;" | Enzyme | |||
! width="50" style="background:#efefef;" | ''Avr'' II | |||
! width="50" style="background:#efefef;" | ''Bam'' HI | |||
! width="50" style="background:#efefef;" | ''Bgl'' II | |||
! width="50" style="background:#efefef;" | ''Eco'' RI | |||
! width="50" style="background:#efefef;" | ''Eco'' RV | |||
! width="50" style="background:#efefef;" | ''Hind'' III | |||
! width="30" style="background:#efefef;" | ''Kpn'' I | |||
! width="30" style="background:#efefef;" | ''Nde'' I | |||
! width="30" style="background:#efefef;" | ''Pst'' I | |||
! width="30" style="background:#efefef;" | ''Sac'' I | |||
! width="30" style="background:#efefef;" | ''Sal'' I | |||
! width="50" style="background:#efefef;" | ''Sma'' I | |||
! width="30" style="background:#efefef;" | ''Spe'' I | |||
! width="30" style="background:#efefef;" | ''Sph'' I | |||
! width="30" style="background:#efefef;" | ''Xba'' I | |||
|- | |||
| '''''Bam'' HI''' | |||
| seq<sup><font color=red>1</font></sup> | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
|- | |||
| '''''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<sup><font color=red>2</font></sup> | |||
| 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 | |||
|- | |||
|} | |||
<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. | |||
<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.