Template:SBB-Protocols Enz2: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
JCAnderson (talk | contribs) |
No edit summary |
||
| Line 4: | Line 4: | ||
8uL of eluted PCR product | 8uL of eluted PCR product | ||
1uL of NEB Buffer 2 | 1uL of NEB Buffer 2 | ||
1uL EcoRI | |||
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 16:52, 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
