Template:SBB-Protocols Enz1

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(EcoRI/BamHI Digest of Wobble Products)
Current revision (19:57, 23 June 2010) (view source)
 
Line 4: Line 4:
   50uL eluted DNA
   50uL eluted DNA
   5.7uL NEB Buffer 2
   5.7uL NEB Buffer 2
-
   1uL EcoRI
+
   0.5uL EcoRI
-
   1uL BamHI
+
   0.5uL BamHI
*Mix thoroughly by slamming the tube upside down on the table, and then a quick spin to move the liquid to the bottom of the tube.
*Mix thoroughly by slamming the tube upside down on the table, and then a quick spin to move the liquid to the bottom of the tube.
*Incubate the reaction at 37 degrees on the thermocycler
*Incubate the reaction at 37 degrees on the thermocycler
*Proceed to another Zymo small fragment cleanup
*Proceed to another Zymo small fragment cleanup

Current revision

EcoRI/BamHI Digest of Wobble Products

For wobble products, you will digest the entire extension reaction-worth of DNA Since you started with 50uL of extension reaction, you should now have 50uL of eluted DNA in water.

  • Set up the following reaction in a PCR tube:
 50uL eluted DNA
 5.7uL NEB Buffer 2
 0.5uL EcoRI
 0.5uL BamHI
  • Mix thoroughly by slamming the tube upside down on the table, and then a quick spin to move the liquid to the bottom of the tube.
  • Incubate the reaction at 37 degrees on the thermocycler
  • Proceed to another Zymo small fragment cleanup
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