Griffitts:Restriction digest

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m (15 μL reaction)
m (30 μL reaction)
Line 22: Line 22:
===30 μL reaction===
===30 μL reaction===
* 12 μL DNA
* 12 μL DNA
-
* 4.3 μL appropriate 10X buffer
+
* 3.3 μL appropriate 10X buffer
-
* 4.0 μL 10X BSA (if needed--see below)
+
* 3.0 μL 10X BSA (if needed--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 30 μL
* Add ddH<sub>2</sub>0 to bring the final volume to 30 μL
Note: Use this recipe when your DNA concentrations are sufficient
Note: Use this recipe when your DNA concentrations are sufficient
 +
===40 μL reaction===
===40 μL reaction===
* 20 μL DNA
* 20 μL DNA

Revision as of 15:24, 16 January 2008

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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
  • 1.8 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
  • 3.3 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

40 μL reaction

  • 20 μL DNA
  • 4.3 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

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 1001 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

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 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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