Griffitts:Restriction digest

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(Enzymes)
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! width="30" style="background:#efefef;" | Enzyme
! width="30" style="background:#efefef;" | Enzyme
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! width="90" style="background:#efefef;" | Amount
+
! width="30" style="background:#efefef;" | Amount
! width="90" style="background:#efefef;" | Target
! width="90" style="background:#efefef;" | Target
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! width="90" style="background:#efefef;" | Overhang
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! 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
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! 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
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! width="60" style="background:#efefef;" | T°
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! width="30" style="background:#efefef;" | T°
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! width="60" style="background:#efefef;" | BSA
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! width="30" style="background:#efefef;" | BSA
|-
|-
-
| ''Avr''II
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| ''Avr'' II
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| c/ctagg
| c/ctagg
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| No
| No
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|-
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| ''Bam''HI
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| ''Bam'' HI
| 1 μL
| 1 μL
| g/gatcc
| g/gatcc
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| 50
| 50
| 75
| 75
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| ''Bam''HI
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| '''''Bam'' HI'''
| 37°C
| 37°C
| Yes
| Yes
|-
|-
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| ''Bgl''II
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| ''Bgl'' II
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| a/gatct
| a/gatct
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| No
| No
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| ''Bst''XI
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| ''Bst'' XI
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| ccannnnn/ntgg
| ccannnnn/ntgg
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| No
| No
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|-
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| ''Eco''RI
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| ''Eco'' RI
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| g/aattc
| g/aattc
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| 100
| 100
| 100
| 100
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| ''Eco''RI
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| '''''Eco'' RI'''
| 37°C
| 37°C
| No
| No
|-
|-
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| ''Eco''RV
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| ''Eco'' RV
|
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| gat/atc
| gat/atc
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| Yes
| Yes
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|-
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| ''Hind''III
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| ''Hind'' III
|
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| a/agctt
| a/agctt
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| No
| No
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|-
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| ''Kpn''I
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| ''Kpn'' I
|
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| ggtac/c
| ggtac/c
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| Yes
| Yes
|-
|-
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| ''Nde''I
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| ''Nde'' I
|
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| ca/tatg
| ca/tatg
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| No
| No
|-
|-
-
| ''Pst''I
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| ''Pst'' I
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| ctgca/g
| ctgca/g
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| Yes
| Yes
|-
|-
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| ''Sac''I
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| ''Sac'' I
|
|
| gagct/c
| gagct/c
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| Yes
| Yes
|-
|-
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| ''Sal''I
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| ''Sal'' I
|
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| g/tcgac
| g/tcgac
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| Yes
| Yes
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|-
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| ''Sma''I
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| ''Sma'' I
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| ccc/ggg
| ccc/ggg
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| No
| No
|-
|-
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| ''Spe''I
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| ''Spe'' I
|
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| a/ctagt
| a/ctagt
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| Yes
| Yes
|-
|-
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| ''Sph''I
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| ''Sph'' I
|
|
| gcatg/c
| gcatg/c
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| No
| No
|-
|-
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| ''Xba''I
+
| ''Xba'' I
| 1.5 μL
| 1.5 μL
| t/ctaga
| t/ctaga
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| Yes
| Yes
|-
|-
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| ''Xho''I
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| ''Xho'' I
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| c/tcgag
| c/tcgag
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''Adapted from New England BioLabs 2005-06 Catalog and Technical Reference.''

Revision as of 18:15, 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.

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