Griffitts:Restriction digest

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Created page)
Current revision (14:38, 20 March 2012) (view source)
(Enzymes)
 
(26 intermediate revisions not shown.)
Line 1: Line 1:
-
{{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)
-
==Recipe==
+
 
-
* 12-20 μL DNA depending on concentration
+
==Recipes==
-
* 4.3 μL appropriate 10X buffer
+
===15 μL reaction===
-
* 4.0 μL 10X BSA
+
* 5 μL DNA
 +
* 1.5 μL appropriate 10X buffer
 +
* 1.5 μL 10X BSA (if needed&mdash;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&mdash;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&ndash;''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&mdash;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&ndash;''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="90" style="background:#efefef;" | Amount
+
! width="30" style="background:#efefef;" | Amount
! width="90" style="background:#efefef;" | Target
! width="90" style="background:#efefef;" | Target
-
! width="90" style="background:#efefef;" | Overhang
+
! 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
! width="60" style="background:#efefef;" | NEBuffer3
! width="60" style="background:#efefef;" | NEBuffer3
! width="60" style="background:#efefef;" | NEBuffer4
! width="60" style="background:#efefef;" | NEBuffer4
 +
! width="60" style="background:#efefef;" | Other Buffer
 +
! width="30" style="background:#efefef;" | T°
 +
! 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
 +
| None
 +
| 37°C
 +
| No
|-
|-
-
| ''Bam''HI
+
| ''Bam'' HI
-
| 1 μL
+
| 0.8 μL
| g/gatcc
| g/gatcc
| gatc
| gatc
Line 50: Line 84:
| 50
| 50
| 75
| 75
 +
| '''''Bam'' HI'''
 +
| 37°C
 +
| Yes
|-
|-
-
| ''Bgl''II
+
| ''Bgl'' II
|
|
| a/gatct
| a/gatct
Line 59: Line 96:
| '''100'''
| '''100'''
| 10
| 10
 +
| None
 +
| 37°C
 +
| No
|-
|-
-
| ''Bst''XI
+
| ''Bst'' XI
|
|
| ccannnnn/ntgg
| ccannnnn/ntgg
Line 68: Line 108:
| '''100'''
| '''100'''
| 50
| 50
 +
| None
 +
| 55°C
 +
| No
|-
|-
-
| ''Eco''RI
+
| ''Eco'' RI
|
|
| g/aattc
| g/aattc
Line 77: Line 120:
| 100
| 100
| 100
| 100
 +
| '''''Eco'' RI'''
 +
| 37°C
 +
| No
|-
|-
-
| ''Eco''RV
+
| ''Eco'' RV
|
|
| gat/atc
| gat/atc
Line 86: Line 132:
| '''100'''
| '''100'''
| 50
| 50
 +
| None
 +
| 37°C
 +
| Yes
|-
|-
-
| ''Hind''III
+
| ''Hind'' III
|
|
| a/agctt
| a/agctt
Line 95: Line 144:
| 10
| 10
| 50
| 50
 +
| None
 +
| 37°C
 +
| No
|-
|-
-
| ''Kpn''I
+
| ''Kpn'' I
|
|
| ggtac/c
| ggtac/c
Line 104: Line 156:
| 0
| 0
| 75
| 75
 +
| None
 +
| 37°C
 +
| Yes
|-
|-
-
| ''Nde''I
+
| ''Nde'' I
|
|
| ca/tatg
| ca/tatg
Line 113: Line 168:
| 75
| 75
| '''100'''
| '''100'''
 +
| None
 +
| 37°C
 +
| No
|-
|-
-
| ''Pst''I
+
| ''Pst'' I
|
|
| ctgca/g
| ctgca/g
Line 122: Line 180:
| '''100'''
| '''100'''
| 50
| 50
 +
| None
 +
| 37°C
 +
| Yes
|-
|-
-
| ''Sac''I
+
| ''Sac'' I
|
|
| gagct/c
| gagct/c
Line 131: Line 192:
| 10
| 10
| 100
| 100
 +
| None
 +
| 37°C
 +
| Yes
|-
|-
-
| ''Sal''I
+
| ''Sal'' I
|
|
| g/tcgac
| g/tcgac
Line 140: Line 204:
| '''100'''
| '''100'''
| 0
| 0
 +
| None
 +
| 37°C
 +
| Yes
|-
|-
-
| ''Sma''I
+
| ''Sma'' I
|
|
| ccc/ggg
| ccc/ggg
Line 149: Line 216:
| 0
| 0
| '''100'''
| '''100'''
 +
| None
 +
| 25°C
 +
| No
|-
|-
-
| ''Spe''I
+
| ''Spe'' I
|
|
| a/ctagt
| a/ctagt
Line 158: Line 228:
| 25
| 25
| 75
| 75
 +
| None
 +
| 37°C
 +
| Yes
|-
|-
-
| ''Sph''I
+
| ''Sph'' I
|
|
| gcatg/c
| gcatg/c
Line 167: Line 240:
| 50
| 50
| 100
| 100
 +
| None
 +
| 37°C
 +
| No
|-
|-
-
| ''Xba''I
+
| ''Xba'' I
| 1.5 μL
| 1.5 μL
| t/ctaga
| t/ctaga
Line 175: Line 251:
| '''100'''
| '''100'''
| 75
| 75
-
| 75
+
| '''100'''
 +
| None
 +
| 37°C
 +
| Yes
|-
|-
-
| ''Xho''I
+
| ''Xho'' I
|
|
| c/tcgag
| c/tcgag
Line 185: Line 264:
| 100
| 100
| 100
| 100
 +
| None
 +
| 37°C
 +
| Yes
 +
|-
 +
|}
 +
<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&ndash;''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.''

Current revision

Restriction Digest

Contents

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

Restriction Enzymes
Enzyme Amount Target Overhang NEBuffer1 NEBuffer2 NEBuffer3 NEBuffer4 Other Buffer 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

Suggested Buffers
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.

Personal tools