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Restriction Map/ Restriction Digest
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==Restriction Map Design== | ==Restriction Map Design== | ||
| - | Restriction maps are designed through Vector NTI plasmid sequences. Restriction sites with compatible enzymes (similar temperatures and buffers needed) are | + | Restriction maps are designed through Vector NTI plasmid sequences. Restriction sites with compatible enzymes (similar temperatures and buffers needed) are identified, and those enzymes are then used to digest the vector plasmid. The restriction site must also be in the proper area of the plasmid for insertion of other necessary isolated DNA. The inserted DNA must also have the cut sites, so areas of DNA will overlap when the plasmid is ligated. |
==Restriction Digest Protocol== | ==Restriction Digest Protocol== | ||
Latest revision as of 01:00, 30 October 2008
PRINCETON IGEM 2008
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Restriction Map Design
Restriction maps are designed through Vector NTI plasmid sequences. Restriction sites with compatible enzymes (similar temperatures and buffers needed) are identified, and those enzymes are then used to digest the vector plasmid. The restriction site must also be in the proper area of the plasmid for insertion of other necessary isolated DNA. The inserted DNA must also have the cut sites, so areas of DNA will overlap when the plasmid is ligated.
Restriction Digest Protocol
Calculations
Divide the OD of the DNA by a suitable number, depending on concentration. If it is a high concentration, divide by 1000. If not, then something such as 750 will also work. Do not use concentrations that are too low, however, because there will not be enough DNA.
Judging from this number, choose a round number (increasing by 5s usually works best) for your total volume that will allow the next steps to be completed succesfully, without an excessive amount of water being added.
The amount of enzyme and buffer should be the same, at 10% of the TOTAL value.
IF BSE is added, that should be 10% of the amount of enzyme.
Use autoclaved water to add the rest of the volume until the total amount is reached.
Digestion and Running on Gel
Place in machine on "Digestion" Cycle for 3hrs.
Run on gel, analyze for correct bands based on restriction map
