Annealing primers

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
Line 15: Line 15:
*Unless you have ordered your primers with 5' phosphate added you will probably improve the efficiency of any subsequent cloning steps by using adding the 5' phosphate using a protocol such as [[PNK Treatment of DNA Ends]]
*Unless you have ordered your primers with 5' phosphate added you will probably improve the efficiency of any subsequent cloning steps by using adding the 5' phosphate using a protocol such as [[PNK Treatment of DNA Ends]]
 +
 +
*If you are interested in cloning piece of DNA and include restriction enzyme recognition sites near the ends of the linear DNA fragment, note that some enzymes do not cut efficiently at the ends of linear DNA.  So include extra bases to increase the efficiency of cutting.  Many enzymes work with 4 bases supposedly but XhoI was found to require more than 4 bases (8 bases was used successfully).  Thus, to be on the safe side, use 8 bases whenever possible. [http://www.neb.com/ NEB] has more information [http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/cleavage_linearized_vector.asp here].  Read the information at NEB carefully ... they recommend adding 4 bases to the numbers listed in their table.

Revision as of 13:31, 11 May 2005

A simple and cheap way to make a short (<100bp) piece of DNA is to order two complementary primers from a company such as Invitrogen.

  • When the primers arrive, redissolve them in 50mM Tris buffer to yield a concentration of ~800ng/μl.
  • For the annealing mix one recipe that works is as follows -
    • 4μl of each of the concentrated primers.
    • 4μl of salt solution (10mM NaCl)
    • 28μl of water
  • The salt shields the negative charges on the single-stranded DNA molecules, allowing them to come close enough to bind.
  • Anneal the primers by heating them at least 5oC above their melting point and cooling them down slowly in stages using a Thermocycler. Melting Temperature calculations can best be done using software such as VectorNTI or data may come with the primers themselves.
  • A simpler approach is to add the above mix in a PCR tube to a beaker of boiling water and just allow the water to cool down naturally. Most primers pairs with length less than 100bp should be fully melted at 100oC and certainly any non-specific binding should be melted at that temperature.
  • Unless you have ordered your primers with 5' phosphate added you will probably improve the efficiency of any subsequent cloning steps by using adding the 5' phosphate using a protocol such as PNK Treatment of DNA Ends
  • If you are interested in cloning piece of DNA and include restriction enzyme recognition sites near the ends of the linear DNA fragment, note that some enzymes do not cut efficiently at the ends of linear DNA. So include extra bases to increase the efficiency of cutting. Many enzymes work with 4 bases supposedly but XhoI was found to require more than 4 bases (8 bases was used successfully). Thus, to be on the safe side, use 8 bases whenever possible. NEB has more information here. Read the information at NEB carefully ... they recommend adding 4 bases to the numbers listed in their table.
Personal tools