Help me! How to design nested BSP primers? - (Aug/14/2009 )

PCRMAN and methynick, please help.
I am now doing nested BSP, but i don't know how to design nested BSP primer, can you take an example to explain nested BSP primer design? So many people are willing to know the method, it is kindhearted for you to do so!

Primer design is the same as those notes that I have posted and pinned to the forum. Same criteria,

for a nested set you are designing a pair of internal primers to the original ones for your region of interest, then your perform two rounds of PCR, the first round with your outer primer set, then take an aliquot of that into your second round PCR using your internal primer set.

Easy as

I tend to do hemi nested PCRs so I have one internal primer and repeat the second round with that and the opposing first round primer.

hope this explains things

Thanks for your reply, but i can't really know the proccedure of primer design as you told me, you mean design one pair primer and then design another one(inner)? which software do you use? methyl express? I really hope that you take an example to illustrate the process! Thx again for your kind reply!

I design manaully without a software package, you could use methyl primer express.

Nick

Thanks for your reply,i still have one questions,netsted BSP:1st set primers for original DNA sequence or modified sequence? second set primers for what?