8 replies to this topic

[ipman_81]

For over a month now I've been trying to get Fusion / Overlap PCR to work. The protocol designed by my lab is robust and has always worked for everyone in the past. I've made new reagents, redesigned primers, tried different enzymes and I still can't seem to make it work efficiently. There are some bands that seem to be the right size, but they are not the prominent bands on gel. The fragments for this Fusion PCR are constructed with very little trouble, but this last step is not working.

Can anyone shed some light or give me some tips on how I should go about tackling this issue? I'm trying not to stray too far away from the labs robust protocol, but at this point I'm willing to try anything to make it work.

[OA17]

- Have you checked that PCR program that you are using has an annealing temperature suitable for your ovelapping sequences, an not only for external primers?

- In some cases, I have heard that doing some initial PCR cycles without adding primers to the reaction (with the right annealing temperature, so that the two ovelapping sequences can anneal), and then adding the primers and doing the normal PCR cycles can help.

- Another option in order to increase the specificity could be to do a touch-down PCR.

- I normally use DNA templates that overlap in around 20 bases. If our overlapping sequence is shorter, then it may anneal to other non-desired region in your template, and that's why you get unspecific bands.

- Check that you are putting the right amount of each DNA template and that these have been well purified (i.e. free of PCR inhibitors, etc...)

Good luck!! :-)

[Curtis]

here, read this:
http://www.protocol-...pcr#entry139028

[ipman_81]

Well the method of not adding primers until a few cycles didn't work. I thought that was a really good idea though. Thank you OA17.

[OA17]

You're welcome! Good luck, anyway! :-)

[ipman_81]

Ok so now I'm really sad. The Fusion PCR reaction is working very efficiently in other peoples hands in the lab. What could I be doing wrong that everyone is doing right?

[Curtis]

Not enough template.use excess

[OA17]

Is this same PCR working in other people's hands and not in yours, or are they doing different PCRs?

If it's the same PCR, maybe you are not doing a correct master mix. For example, you could be adding too many dNTPs, etc...
Check that the concentration of your reagents is o.k. and if I were you, I would also check that the primers are in good condition.

[ipman_81]

Finally got it to work like everyone else's!! Ok so it seems as though the mid-piece had some issues. I confirmed the primer sequence for amplifying the midpiece and did an experiment to test between what I had been using this whole time and a newly amplified midpiece fragment. The new ones came out almost flawless! =)

So I think the lesson is that even if people in the lab have done it a thousand times, make sure you understand the method thoroughly and double check everything that is handed down to you.