Help:Protocol/DNA Synthesis


Constructing a BioBrick part via direct synthesis=

If you are constructing a BioBrick part via direct synthesis ... especially a coding sequence (in which the sequence is flexible), you may want to consider eliminating the following restriction sites that might be useful in the future to others. This list of sites is prioritized.

These sites MUST be absent

The exception to this rule is for the BioBricks prefixes, BioBricks suffixes and the multiple cloning site of BioBricks vectors.

Strongly prefer that these sites be absent

These restriction enzymes are those that generate compatible cohesive ends to the BioBrick sites and therefore can be useful for various projects.

  • [http://www.neb.com/nebecomm/products/productR0566.asp ApoI] (probably impossible because it occurs frequently), [http://www.neb.com/nebecomm/products/productR0589.asp Mfe I]
  • [http://www.neb.com/nebecomm/products/productR0174.asp AvrII], [http://www.neb.com/nebecomm/products/productR0131.asp NheI]
  • [http://www.neb.com/nebecomm/products/productR0127.asp NsiI], [http://www.neb.com/nebecomm/products/productR0642.asp SbfI]

These restriction sites are used in other assembly standards

  • BamHI, BglII, XhoI (Berkeley standard)
  • NgoMIV, AgeI (Freiburg standard)

Would prefer that these sites be absent

Offset cutters

These are offset cutters that can generate arbitrary overhangs. In addition, some are used in the BioBricks++ assembly scheme.

  • [http://www.fermentas.com/catalog/re/aari.htm AarI], [http://www.neb.com/nebecomm/products/productR0641.asp AcuI] (medium priority), [http://www.neb.com/nebecomm/products/productR0539.asp BbsI], [http://www.neb.com/nebecomm/products/productR0601.asp BbvCI], [http://www.neb.com/nebecomm/products/productR0596.asp BciVI] (would be nice, medium priority), [http://www.neb.com/nebecomm/products/productR0701.asp BfuAI] (high priority), [http://www.neb.com/nebecomm/products/productR0600.asp BmrI] (high priority), [http://www.neb.com/nebecomm/products/productR0580.asp BsmBI], [http://www.neb.com/nebecomm/products/productR0535.asp BsaI], [http://www.neb.com/nebecomm/products/productR0559.asp BsgI] (medium priority), [http://www.neb.com/nebecomm/products/productR0134.asp BsmI] (includes nicking enzyme, high priority), [http://www.neb.com/nebecomm/products/productR0502.asp BspMI], [http://www.neb.com/nebecomm/products/productR0574.asp BsrDI] (includes nicking enzyme, high priority), [http://www.neb.com/nebecomm/products/productR0703.asp BtgZI], [http://www.neb.com/nebecomm/products/productR0528.asp EarI], [http://www.neb.com/nebecomm/products/productR0646.asp EcoP15I] (high priority), [http://www.neb.com/nebecomm/products/productR0109.asp FokI] (best effort), [http://www.neb.com/nebecomm/products/productR0607.asp Nt.BstNBI], [http://www.neb.com/nebecomm/products/productR0569.asp SapI] (high priority, should already be eliminated from EarI), [http://www.neb.com/nebecomm/products/productR0582.asp TspRI] (probably difficult, best effort)

Nicking enzymes

  • [http://www.neb.com/nebecomm/products/productR0607.asp Nt.BstNBI], BbvCI, [http://www.neb.com/nebecomm/products/productR0627.asp Nt.AlwI] (best effort to at least remove sites near each other)

Homing endonucleases

These enzymes have very long recognition sites and are unlikely to be in your part.

  • I-CeuI, I-SceI, PI-PspI, PI-SceI, I-PpoI

Others

  • [http://www.neb.com/nebecomm/products/productR0552.asp AgeI], AscI, FseI, [http://www.neb.com/nebecomm/products/productR0501.asp RsrII], [http://www.neb.com/nebecomm/products/productR0603.asp SgrAI], [http://www.neb.com/nebecomm/products/productR0194.asp XmnI], [http://www.neb.com/nebecomm/products/productR0533.asp XcmI]

Would be convenient if these sites were removed

These are common, efficient cutters that people might want to use.

  • HindIII, BamHI, XhoI, NcoI, SacI, NdeI

Here are other low priority cut sites to remove

  • KasI, MssI, NgoMIV, PacI, PmeI, SalI, SfiI, SgfI, SmiI, SrfI, SwaI, XmaI, ZraI

Sites to include

  • Having GATC sites in your part can sometimes be useful because it allows plasmid purified DNA to be cut (via DpnI) whereas PCR product DNA is not. Such a trick is useful for some site directed mutagenesis protocols.

Miscellaneous

  • BaeI is an enzyme whose is exact cut position is unknown. It is explicitly included in some pSB plasmid replications origins so that the plasmid can optionally be destroyed via another enzyme. You may want to eliminate this site from your part if you intend to use this feature of the BioBricks plasmids.

Source: http://openwetware.org/wiki/Synthetic_Biology:BioBricks/Part_fabrication