Griffitts:Restriction digest

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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
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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