Hi Matin

I would not worry too much about things like penalty scores in Primer 3: Primer 3 is a robust package and defaults of these and other things tend to be set for reproducible results:

There are however things to consider in terms of optimal primer design and I have explained these in an SOP on good primer design (attached)

In particular I would:

1. Set your primer design parameters so that primers differ by no more than 2C in terms of predicted melting temp

2. Consider the composition of the last 5bp in the primer (in particular). Specifically, avoid terminating with double T's and A's (which reduced priming efficiency) and > 3 consecutive G/C residues at the 3' or 5' terminus (which increases the likely hood of primer dimers and other duplex structures

3. Do however incorporate 3 G/C residues in the last 5 bases @ the 3' terminus (which maximises priming efficiency)  and terminate with 2 x G/C residues (although 1 x G or C residue acceptable; just not 3 !!)

4. Finally, when computing the respective Tm's of your primers try and reflect actual PCR conditions in terms of primer concentration; Mg concentration and dNTP concentration as these have an effect on duplex stability and hence Tm in your actual PCR reaction

When using primer 3 I tend to look for intra prier binding using the following program

http://biotools.nubic.northwestern.edu/OligoCalc.html

And check for inter (hetero) F:R primer dimers as well as calculating Tms in the context of simulated PCR reactions using the following program:

http://www.idtdna.com/calc/analyser

Hi,

There will be only one primer in your sequencing reaction. Therefore, the penalty score of the pair is not important in this experiment.

Are you planning a amplification by PCR prior to sequencing? In that case, you could increase the distance of your primer binding sites to the point mutation. There could be better primers in the more distant regions. However, even primers which failed the quality control of Primer3 can work in the wet lab. Try to keep the penalty score as low as possible.

Good luck!

Hi Matin

I would not worry too much about things like penalty scores in Primer 3: Primer 3 is a robust package and defaults of these and other things tend to be set for reproducible results:

There are however things to consider in terms of optimal primer design and I have explained these in an SOP on good primer design (attached)

In particular I would:

1. Set your primer design parameters so that primers differ by no more than 2C in terms of predicted melting temp

2. Consider the composition of the last 5bp in the primer (in particular). Specifically, avoid terminating with double T's and A's (which reduced priming efficiency) and > 3 consecutive G/C residues at the 3' or 5' terminus (which increases the likely hood of primer dimers and other duplex structures

3. Do however incorporate 3 G/C residues in the last 5 bases @ the 3' terminus (which maximises priming efficiency)  and terminate with 2 x G/C residues (although 1 x G or C residue acceptable; just not 3 !!)

4. Finally, when computing the respective Tm's of your primers try and reflect actual PCR conditions in terms of primer concentration; Mg concentration and dNTP concentration as these have an effect on duplex stability and hence Tm in your actual PCR reaction

When using primer 3 I tend to look for intra prier binding using the following program

http://biotools.nubic.northwestern.edu/OligoCalc.html

And check for inter (hetero) F:R primer dimers as well as calculating Tms in the context of simulated PCR reactions using the following program:

http://www.idtdna.com/calc/analyser