long PCR primer - (Mar/17/2009 )

hello,
I plan to cut out a fragment from a plasmid and then amplify this fragment with PCR.
In addition to amplify the fragment, i also want to add something behind this fragment to modify it. and then ligate it to another plasmid.

I read a paper that used a 42bp forward primer and a 106bp reverse primer (BMC Biology 2008, 6:40).
My fragment is 2kb long and the fragment that i want to add behind it is 64bp long. So i designed a 47bp forward primer and a 96bp reverse primer based on this paper.

I am new in the field, but i always heard people say that ~20 to 25bp primers are optimal. SO, i was wondering is it possible to use such long primers to PCR my fragment?? How will it work?? is there any specific point that i have to pay attention while doing long primers PCR???

thank you in advance for your suggestion.

Ah-Do on Mar 17 2009, 05:19 AM said:

Yes it is. I regularly use primers 100bp long. the tm of the primer is based only on the bp that actually bind to the template. I pick a tm around 58C to 63C. A high tm means that the primer is less likely to self anneal and has a higher specificity to the template sequence.

Ah-Do on Mar 17 2009, 05:19 AM said:

It is important to keep an eye out for primer self annealing and hairpin structure formation. I use northwestern oligo calculator to check for tm, hairpin loops and primer self annealing.

perneseblue on Mar 17 2009, 06:47 AM said:

Thank you for ur reply. I check the Tm on the website you mentioned. However, i got very high Tm (>70C~80C) and possibilities of dimer/hairpin formation.

How can i modified my primers regarding lowering the Tm??

the attached file is the schematic description of my question. Thank you again for any advice!!

Ah-Do on Mar 17 2009, 07:36 AM said:

Don't use the entire primer. For tm calculations use only the segment of the primer that actually binds to the template. Once you have a template binding sequence which has the desired tm (eg 58C), then check the completed primer for self annealing.

the schematic description looks okay.

perneseblue on Mar 17 2009, 10:29 AM said:

Thank you!
I've check the Tm based on your advice. The Tm are within acceptable range, but the completed primers still have high potential of self-annealing and hairpin formation. what should i do with it?

Thanks again.

Ah-Do on Mar 17 2009, 12:41 PM said:

Is there any possibility to fix that problem by changing the DNA sequence? If not, you will have to rely on the high tm to melt these problems. If the tm is high (58-62 C), it is often okay.

Frankly, I wouldn't care about hairpins and self-ligation much unless its more than 35%. You can check that using Amplify3X (on Mac, don't know equivalent Windows program)

I agree with whatever perneseblue has mentioned so far, just one suggestion. Try to amplify your sequence right from the plasmid instead of cutting it out. In my experience, PCR on digested DNA fragment may not work all the time. You have nothing to lose even if you run it on the plasmid as your primers will bind to the region of interest and amplify the sequence and during extension phase, it will add up those new nucleotides on your amplified sequence. Does it make sense to you?

noelmathur on Mar 18 2009, 12:37 AM said:

Thank you for your advice.

Actually, i haven't done the PCR with a digested fragment so far. I just simply thought that maybe the primers will bind to regions other than the sequence of interest since the plasmid is very big (~12kb). if i cut out the fragment, the primers won't have chances to bind to other places. It's just my superficial inference. But, i am not very experienced in this, so maybe i will try both ways to see which can give me the best result.

Thank you again for your suggestion.