Restriction Map/ Restriction Digest

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Revision as of 02:53, 29 October 2008

Restriction Map Design

Restriction maps are designed through Vector NTI plasmid sequences. Restriction sites with compatible enzymes (similar temperatures and buffers needed) are found, 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

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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 used) are found, 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 maps are designed through Vector NTI plasmid sequences. Restriction sites with compatible enzymes (similar temperatures and buffers needed) are found, 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==