Verifying the structure of DNA constructs
using restriction digests
Due by 11:59 pm, Friday March 22.
Notes on Restriction
digests 1)Remember that the three rightmost columns in the BACHREST output, with the column headings Frags, Begin and End, give the sizes of fragments seen in descending order of size, as they would appear on a gel, and the beginning and end of each fragment. 2) Some enzymes recognize several possible restriction sequences. For example, SmlI recognizes 5'C^TYRAG3', where Y stands for pyrimidine (C or T) and R stands for purine (A or G). See Sequence File Formats for a complete list of ambiguity symbols for nucleotides. 3) CspCI - If you are considering CspCI, be aware that this enzyme is a special case of an enzyme that has two alternative cutting sites on each strand (see CspCI at REBASE). For most cloning applications, CspCI should probably not be used. Because the REBASE file read by Ugene lists each of the sets of cutting sites as a separate instance of CspCI, Ugene will find twice as many CspCI sites as there are. 4) Size estimation on an agarose gel is not very accurate. Assume that we can't detect differences of less than 10%. |
Note on maps Where two or more restriction sites are very close together, the precise ordering of those sites shown in the map may not be correct, depending on how Ugene draws the map. |
fixed font |
#
of Enzyme Recognition Sequence Sites Sites Frags Begin End ------------------------------------------------------------------------------ AcoI Y^CCGGR 3 1289 2817 2533 5349 1556 1288 1 1288 2533 977 1556 2532 267 1289 1555 |
proportional font |
AcoI
Y^CCGGR
3 1289 2817 2533 5349 1556 1288 1 1288 2533 977 1556 2532 267 1289 1555 |