Griffitts:Restriction digest: Difference between revisions

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{{Griffitts}}
<div class="noprint">
{{Template:Griffitts}}
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<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 41: Line 69:
| ctag
| ctag
| 100
| 100
| '''100'''
| '''100'''<sup>1</sup>
| 50
| 50
| 100
| 100
Line 49: Line 77:
|-
|-
| ''Bam'' HI
| ''Bam'' HI
| 1 μL
| 0.8 μL
| g/gatcc
| g/gatcc
| gatc
| gatc
Line 223: Line 251:
| '''100'''
| '''100'''
| 75
| 75
| 75
| '''100'''
| None
| None
| 37°C
| 37°C
Line 241: Line 269:
|-
|-
|}
|}
<sup>1</sup>'''Boldface''' indicates the preferred buffer for the enzyme.
<br><br>
<br><br>
''Adapted from New England BioLabs 2005-06 Catalog and Technical Reference.''
''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.''

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

Restriction 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

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.

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