Template:SBB-Protocols Enz2

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(EcoRI/BamHI Digest of PCR Products)
Line 4: Line 4:
   8uL of eluted PCR product
   8uL of eluted PCR product
   1uL of NEB Buffer 2
   1uL of NEB Buffer 2
-
   0.5uL EcoRI
+
   1uL EcoRI
-
   0.5uL BamHI
+
   1uL BamHI
*Incubate at 37 degrees on the thermocycler for 1hr
*Incubate at 37 degrees on the thermocycler for 1hr
*Run an agarose gel, and melt with 600uL ADB buffer at 55 degrees.  ****NOTE:  If you are running short of time, this is an acceptable stopping point
*Run an agarose gel, and melt with 600uL ADB buffer at 55 degrees.  ****NOTE:  If you are running short of time, this is an acceptable stopping point
*If the DNA is shorter than 300bp, add 250uL of isopropanol and mix prior to loading it on the column
*If the DNA is shorter than 300bp, add 250uL of isopropanol and mix prior to loading it on the column

Revision as of 19:52, 23 June 2010

EcoRI/BamHI Digest of PCR Products

For PCR products, you will only digest a portion of your purified PCR product. Note that you must make a minor modification to the procedure if your DNA is shorter than 300bp (you add a little isopropanol to the ADB after melting).

  • Set up the following reaction:
 8uL of eluted PCR product
 1uL of NEB Buffer 2
 1uL EcoRI
 1uL BamHI
  • Incubate at 37 degrees on the thermocycler for 1hr
  • Run an agarose gel, and melt with 600uL ADB buffer at 55 degrees. ****NOTE: If you are running short of time, this is an acceptable stopping point
  • If the DNA is shorter than 300bp, add 250uL of isopropanol and mix prior to loading it on the column
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