Designing primers with recognition sites of restriction enzymes

[butters]

SEQUENCE:
5'- CCG GGA TCC CGC GCG AGG ATG AGT TAT ACT GTC GGT AC -3'
COMPLEMENT:
5'- GTA CCG ACA GTA TAA CTC ATC CTC GCG CGG GAT CCC GG -3'
LENGTH: 38
GC CONTENT: 60.5 %
MELT TEMP: 69.3 ºC
MOLECULAR
WEIGHT: 11735.6 g/mole
EXTINCTION
COEFFICIENT: 363500 L/(mole·cm)
nmole/OD260: 2.75
µg/OD260: 32.29

SEQUENCE:
5'- CCC GGT ACC CTA GAG GAG CTT GTT AAC AGG C -3'
COMPLEMENT:
5'- GCC TGT TAA CAA GCT CCT CTA GGG TAC CGG G -3'
LENGTH: 31
GC CONTENT: 58.1 %
MELT TEMP: 65.6 ºC
MOLECULAR
WEIGHT: 9521.2 g/mole
EXTINCTION
COEFFICIENT: 295700 L/(mole·cm)
nmole/OD260: 3.38
µg/OD260: 32.20

Temperature wise it look ok, however still remember the self dimer and hetero dimer? It is not good. Very stable self/hetero dimer.

the problem is your forward primer 5' end. What about just synthesizing one primer?

[obama]

Try to do gradient PCR to optimize your pcr and try different primer concentration

[saranyah]

Thanks for such an elaborate calculation... I'll try re-synthesizing the forward. Should the annealing temperature or time be manipulated?

[saranyah]

What kind of changes should be done in the cycle? Should the time of each cycle be increased/decreased?

[butters]

Originally Posted by saranyah

Thanks for such an elaborate calculation... I'll try re-synthesizing the forward. Should the annealing temperature or time be manipulated?

DON'T re-synthesize the same forwar primer, it have TOO stable forward 5' end homo/heterodimer. If you do have it optimized using your current forward primer do let us now.

[saranyah]

I cant understand what you have said sir? Should I synthesize another primer or try with the same?

[butters]

you should design another forward primer with less G and C on the 5' end. Please do use the link i posted to check for homo/hetero dimer to avoid the same situation from happening again. Do you have any collegue in the same lab who have experience in primer design and can help?

or you could try your luck by optimizing your pcr primer as suggested by Obama if you cannot order another primer. Anybody in your lab with experience with pcr optimization?

[saranyah]

Unfortunately I'm in a bioinformatics lab and my seniors are not exposed to molecular biology work... I'll try optimizing the PCR and also if I'm having fund I'll synthesize a new forward... Thanks for offering ideas....

[butters]

do update us on the progress, especially me as I am really curious what possible experimental parameter can actually remove that kind of strong binding.