Griffitts:Restriction digest

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! width="60" style="background:#efefef;" | NEBuffer3
! width="60" style="background:#efefef;" | NEBuffer3
! width="60" style="background:#efefef;" | NEBuffer4
! width="60" style="background:#efefef;" | NEBuffer4
 +
! width="60" style="background:#efefef;" | Other Buffer
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! width="60" style="background:#efefef;" | T°
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! width="60" style="background:#efefef;" | BSA
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|-
| ''Avr''II
| ''Avr''II
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| 50
| 50
| 100
| 100
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| None
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| 37°C
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| No
|-
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| ''Bam''HI
| ''Bam''HI
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| 50
| 50
| 75
| 75
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| ''Bam''HI
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| 37°C
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| Yes
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| ''Bgl''II
| ''Bgl''II
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| '''100'''
| '''100'''
| 10
| 10
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| None
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| 37°C
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| No
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| ''Bst''XI
| ''Bst''XI
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| '''100'''
| '''100'''
| 50
| 50
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| None
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| 55°C
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| No
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| ''Eco''RI
| ''Eco''RI
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| 100
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| ''Eco''RI
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| 37°C
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| No
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| '''100'''
| '''100'''
| 50
| 50
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| None
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| 37°C
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| Yes
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| ''Hind''III
| ''Hind''III
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| 10
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| None
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| 37°C
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| No
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| ''Kpn''I
| ''Kpn''I
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| 0
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| None
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| 37°C
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| Yes
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| ''Nde''I
| ''Nde''I
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| '''100'''
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| None
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| 37°C
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| No
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| ''Pst''I
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| '''100'''
| '''100'''
| 50
| 50
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| None
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| 37°C
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| Yes
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| ''Sac''I
| ''Sac''I
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| 10
| 10
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| None
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| 37°C
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| Yes
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| ''Sal''I
| ''Sal''I
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| '''100'''
| '''100'''
| 0
| 0
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| None
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| 37°C
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| Yes
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| ''Sma''I
| ''Sma''I
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| 0
| 0
| '''100'''
| '''100'''
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| None
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| 25°C
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| No
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| ''Spe''I
| ''Spe''I
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| None
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| 37°C
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| Yes
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| ''Sph''I
| ''Sph''I
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| 50
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| None
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| No
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| ''Xba''I
| ''Xba''I
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| 75
| 75
| 75
| 75
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| None
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| 37°C
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| Yes
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| ''Xho''I
| ''Xho''I
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| 100
| 100
| 100
| 100
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| None
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| 37°C
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| Yes
|-
|-
|}
|}

Revision as of 18:02, 8 November 2007

Home     Research     Lab Members     Protocols     Publications     Internal    


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
AvrII c/ctagg ctag 100 100 50 100 None 37°C No
BamHI 1 μL g/gatcc gatc 75 100 50 75 BamHI 37°C Yes
BglII a/gatct gatc 10 75 100 10 None 37°C No
BstXI ccannnnn/ntgg nnnn 25 100 100 50 None 55°C No
EcoRI g/aattc aatt 100 100 100 100 EcoRI 37°C No
EcoRV gat/atc none 50 75 100 50 None 37°C Yes
HindIII a/agctt agct 50 100 10 50 None 37°C No
KpnI ggtac/c gtac 100 75 0 75 None 37°C Yes
NdeI ca/tatg ta 75 100 75 100 None 37°C No
PstI ctgca/g tgca 75 75 100 50 None 37°C Yes
SacI gagct/c agct 100 50 10 100 None 37°C Yes
SalI g/tcgac tcga 0 0 100 0 None 37°C Yes
SmaI ccc/ggg none 0 0 0 100 None 25°C No
SpeI a/ctagt ctag 75 100 25 75 None 37°C Yes
SphI gcatg/c catg 100 100 50 100 None 37°C No
XbaI 1.5 μL t/ctaga ctag 0 100 75 75 None 37°C Yes
XhoI c/tcgag tcga 75 100 100 100 None 37°C Yes
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