Skatebro:Primer Design

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(Tips)
(Tips)
Line 5: Line 5:
*Insert a double stop codon (taa taa)
*Insert a double stop codon (taa taa)
*Binding length of 18-22 bps is ideal
*Binding length of 18-22 bps is ideal
-
*Melt temps. of forward and reverse primers should be within ~2c and ~60c is desirable
+
*Melt temps. of forward and reverse primers should be within ~2c and ~40-60c is desirable
 +
**analyze the landing sequence and the landing+tail Tm (can gradually increase annealing temp over pcr cycles if necessary)
*Make sure primer can only form short/thermodynamically unstable hairpins
*Make sure primer can only form short/thermodynamically unstable hairpins

Revision as of 04:14, 7 February 2008

Skatebro:Project08

Tips

  • In general, want high GC content, up to 50%
  • Insert a double stop codon (taa taa)
  • Binding length of 18-22 bps is ideal
  • Melt temps. of forward and reverse primers should be within ~2c and ~40-60c is desirable
    • analyze the landing sequence and the landing+tail Tm (can gradually increase annealing temp over pcr cycles if necessary)
  • Make sure primer can only form short/thermodynamically unstable hairpins
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