Template:SBB-Protocols Enz2
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Current revision (15:51, 8 February 2011) (view source) |
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===EcoRI/BamHI Digest of PCR Products=== | ===EcoRI/BamHI Digest of PCR Products=== | ||
| - | '''For PCR products, you will only digest a portion of your purified PCR product | + | '''For PCR products, you will only digest a portion of your purified PCR product.''' |
*Set up the following reaction: | *Set up the following reaction: | ||
8uL of eluted PCR product | 8uL of eluted PCR product | ||
1uL of NEB Buffer 2 | 1uL of NEB Buffer 2 | ||
| - | + | 0.5uL EcoRI | |
| - | + | 0.5uL 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 | ||
Current revision
EcoRI/BamHI Digest of PCR Products
For PCR products, you will only digest a portion of your purified PCR product.
- Set up the following reaction:
8uL of eluted PCR product 1uL of NEB Buffer 2 0.5uL EcoRI 0.5uL 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
