Griffitts:Restriction digest

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(Double Digests: added rows)
(Double Digests: added rows)
Line 263: Line 263:
! width="30" style="background:#efefef;" | ''Sph'' I
! width="30" style="background:#efefef;" | ''Sph'' I
! width="30" style="background:#efefef;" | ''Xba'' I
! width="30" style="background:#efefef;" | ''Xba'' I
-
! width="30" style="background:#efefef;" | ''Xho'' I
 
|-
|-
-
| ''Bam'' HI
+
| '''''Bam'' HI'''
-
| seq
+
| seq<sup>1</sup>
-
| -
+
| -
| -
| -
| -
Line 283: Line 281:
| -
| -
|-
|-
-
| ''Bgl'' II
+
| '''''Bgl'' II'''
| 3
| 3
| ''Bam'' HI
| ''Bam'' HI
-
| -
 
| -
| -
| -
| -
Line 301: Line 298:
| -
| -
|-
|-
 +
| '''''Eco'' RI'''
| ''Eco'' RI
| ''Eco'' RI
| ''Eco'' RI
| ''Eco'' RI
| ''Eco'' RI
| ''Eco'' RI
-
| ''Eco'' RI
 
-
| -
 
| -
| -
| -
| -
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| -
| -
|-
|-
-
| ''Eco'' RV
+
| '''''Eco'' RV'''
| 2
| 2
| ''Bam'' HI
| ''Bam'' HI
| 3
| 3
| ''Eco'' RI
| ''Eco'' RI
-
| -
 
| -
| -
| -
| -
Line 337: Line 332:
| -
| -
|-
|-
-
| ''Hind'' III
+
| '''''Hind'' III'''
| 2
| 2
| seq
| seq
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| ''Eco'' RI
| ''Eco'' RI
| 2
| 2
-
| -
 
| -
| -
| -
| -
Line 355: Line 349:
| -
| -
|-
|-
-
| ''Kpn'' I
+
| '''''Kpn'' I'''
| 1
| 1
| seq
| seq
Line 362: Line 356:
| 2
| 2
| 2
| 2
-
| -
 
| -
| -
| -
| -
Line 373: Line 366:
| -
| -
|-
|-
-
| ''Nde'' I
+
| '''''Nde'' I'''
| 4
| 4
| ''Bam'' HI
| ''Bam'' HI
Line 381: Line 374:
| 2
| 2
| 1
| 1
-
| -
 
| -
| -
| -
| -
Line 391: Line 383:
| -
| -
|-
|-
-
| ''Pst'' I
+
| '''''Pst'' I'''
| 3
| 3
| ''Bam'' HI
| ''Bam'' HI
Line 400: Line 392:
| 1
| 1
| 3
| 3
-
| -
 
| -
| -
| -
| -
Line 409: Line 400:
| -
| -
|-
|-
-
| ''Sac'' I
+
| '''''Sac'' I'''
| 1
| 1
| seq
| seq
Line 419: Line 410:
| 4
| 4
| 1
| 1
-
| -
 
| -
| -
| -
| -
Line 427: Line 417:
| -
| -
|-
|-
-
| ''Sal'' I
+
| '''''Sal'' I'''
| seq
| seq
| ''Bam'' HI
| ''Bam'' HI
Line 438: Line 428:
| 3
| 3
| seq
| seq
-
| -
 
| -
| -
| -
| -
Line 445: Line 434:
| -
| -
|-
|-
-
| ''Sma'' I
+
| '''''Sma'' I'''
| 4
| 4
| seq
| seq
Line 457: Line 446:
| 4
| 4
| seq
| seq
-
| -
 
| -
| -
| -
| -
Line 463: Line 451:
| -
| -
|-
|-
-
| ''Spe'' I
+
| '''''Spe'' I'''
| 2
| 2
| ''Bam'' HI
| ''Bam'' HI
Line 479: Line 467:
| -
| -
| -
| -
 +
|-
 +
| '''''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
|-
|-
|}
|}
 +
<sup>1</sup> Sequential digest recommended.
<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.''

Revision as of 19:26, 8 November 2007

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Restriction Digest

Contents

Materials

  • ddH20
  • DNA sample on ice
  • Appropriate 10X buffer on ice
  • 10X BSA on ice
  • Restriction enzyme(s) (see below) on ice
  • CIP (keep in freezer until needed)

Recipe

  • 12-20 μL DNA depending on concentration
  • 4.3 μL appropriate 10X buffer
  • 4.0 μL 10X BSA
  • 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

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

Enzymes

Restriction Enzymes
Enzyme Amount Target Overhang NEBuffer1 NEBuffer2 NEBuffer3 NEBuffer4 Other Buffer BSA
Avr II c/ctagg ctag 100 100 50 100 None 37°C No
Bam HI 1 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



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 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.

Adapted from New England BioLabs 2005-06 Catalog and Technical Reference.

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