Restriction Map
From 2007.igem.org
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Keep the total volume as low as possible. | Keep the total volume as low as possible. | ||
| - | Make gels as before (gel extraction protocol). | + | Make gels as before ([[gel extraction]] protocol). |
Run on the agarose gel for as long as required to obtain maximum resolution. | Run on the agarose gel for as long as required to obtain maximum resolution. | ||
Compare with the expected pattern of bands and pick out the correct try(ies) for maxiprep transformation | Compare with the expected pattern of bands and pick out the correct try(ies) for maxiprep transformation | ||
Latest revision as of 03:08, 27 October 2007
Choose a restriction enzyme such that the enzyme(s) cut both the vector and the insert and the bands can be distinguished reasonably on a gel to identify the correct try. Vector NTI should be used to design restriction maps.
Make sure to create a gel map on vector for the restriction digest. Also remember to digest the parents with the same enzyme(s).
Restriction maps should be setup just like digestions are setup. Carefully calculate the amount of DNA you would need to digest. You need at least 50ng of DNA per band to see it on the gel. The DNA for each band is proportional to its size : for example if you expect to see a 500 bp band and a 4.5 kb out of a 5 kb plasmid after your digest, you need to digest atleast 0.5 µg as they will be divided as 450ng of the 4.5kb band and 50ng of the 500 bp band.
Keep the total volume as low as possible.
Make gels as before (gel extraction protocol).
Run on the agarose gel for as long as required to obtain maximum resolution.
Compare with the expected pattern of bands and pick out the correct try(ies) for maxiprep transformation
