optimization of PCR

Shahram Mori smori at nmsu.edu
Fri Dec 23 18:12:27 EST 1994


Mireille Moulard () wrote:
: Hi everybody!

: Does someone have a valuable reference about PCR optimization
: when using degenerate primers ? My PCR experiments do not work
: and I wonder if I should change my primers or not..
: Any advice is welcome.
: Thanks for your help!

: Mireille
: please forward messages in my box: moulard at evalgb.geneti.uv.es

Dear Mireille,
I don't know how you designed your PCR primers, however it is best to use
those amino acids that have one or 2 codons. The amino acids with one or 2
codons are ( tyr, met, glt, gln, asp, asn, phe, lys,his, cys, trp). Design
primers that are high in these amino acids. Remember the 3' end of the
primer must be very well matched and devoid of Inosine.
Do your annealing at 55C. If too many bands increase a little at a time.
Also the Mg conc. is important.Start 1-2mM Mg and increase in small
increments to 5mM Mg.
Try the following cycle;

95C for 2min
denaturing temp 95C 1min ]
Annealing  temp 55C 1min ]	35-40 Cycles
extention  temp 72  2min ]
final extention 7 min at 72C. 1cycle.  
Hope this helps.
Cheers
--
Shahram Mori					   _/\_
Program in Molecular Biology			  _\  /_
Dept. of chemistry and Biochemistry Box 3C	  \_  _/
NMSU  Las Cruces NM				    ||
88003





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