Griffitts:Restriction digest

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''Adapted from New England BioLabs 2005-06 Catalog and Technical Reference.''
''Adapted from New England BioLabs 2005-06 Catalog and Technical Reference.''
==Double Digests==
==Double Digests==
-
{| border="1" cellpadding="5" cellspacing="0" class="wikitable sortable"
+
{| border="1" cellpadding="5" cellspacing="0"
|+'''Suggested Buffers'''
|+'''Suggested Buffers'''
|-
|-
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! width="30" style="background:#efefef;" | ''Bam'' HI
! width="30" style="background:#efefef;" | ''Bam'' HI
! width="30" style="background:#efefef;" | ''Bgl'' II
! width="30" style="background:#efefef;" | ''Bgl'' II
-
! width="30" style="background:#efefef;" | ''Bst'' XI
 
! width="30" style="background:#efefef;" | ''Eco'' RI
! width="30" style="background:#efefef;" | ''Eco'' RI
! width="30" style="background:#efefef;" | ''Eco'' RV
! width="30" style="background:#efefef;" | ''Eco'' RV
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| ''Bam'' HI
| ''Bam'' HI
| seq
| seq
-
| -
 
| -
| -
| -
| -
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| -
| -
| -
| -
 +
| -
 +
| -
 +
| -
 +
| -
 +
| -
 +
| -
 +
| -
 +
| -
 +
| -
 +
|-
 +
| ''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
| -
| -
| -
| -

Revision as of 18:36, 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 Xho I
Bam HI seq - - - - - - - - - - - - - - -
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 - - - - - - - - - -



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

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