Lost in Fusion PCR - I cannot get any fusion product (Jun/19/2006 )

Hi everyone.I'm completely lost.

I got two templates, one has a overlapping sequence (18bp) to the other one. I try to join them using the templates (100ng each), high fidelity polymerase and a low annealing temperature (48C) for ten cycles. I don't get any higher band. Should I increase my annealing time? Decrease annealing Temp? Use DMSO? Any suggestions?

This is in detail what I've done so far:

I've been trying to achieve this for a while, and after many attempts I come back here to ask for help and advice.

QUOTE (Jiang M @ Mar 7 2006, 08:28 AM)

Lets imagine that the sequence that you want to attach GFP to, is of 2kb for example. GFP sequence thats within the plasmid is of 2kb if I remember correctly. so you can try fusion pCR and then restriction digest. In the sense, design two primers for your insert forward and reverse primer. The end of the sequence where you want to attach GFP to, that will be reverse primer, will be designed in such a way that 18nt bases will act as primer for your sequence and you will attach part of GFP seuquence to this primer, which will actually be forward sequence of GFP primer.
So reverse primer of your insert+GFP forward primer will whole make your reverse primer for insert.

Insert restriction sites at forward primer of your insert and reverse primer of your GFP sequence.

You will amplify GFP as well as insert seperately.

Once both are amplified, check quickly on gel using an aliquot, if you haven't amplified anything else.

Once you see one band, mix portions of both the amplified products and run them in PCR machine with only polymerase for 5 cycles then add forward primer of your construct and reverse primer of GFP sequence and amplify whole stretch to obtain a fusion product.

Once you have this fusion product, cut the plasmid and insert this amplified product there.

QUOTE (del @ Mar 7 2006, 06:37 PM)

It's pretty unlikely you will find an appropriate RE site in your sequence, and there's no need for you to incorporate one just to remove your stop codon - you can do it much more easily by PCR (exactly as described as JiangM). No need for restriction digests, and avoids beard-pulling over ligation problems etc... Just design the downstream fusion-PCR primer for your insert to target the 18bp immediately upstream of the stop codon (don't include it though) then include a sequence 'tag' from the start of the GFP sequence on the 5' end of this primer. The stop codon will be eliminated before you 'fuse' the 2 PCR products.

This is the link:
http://www.protocol-online.org/forums/inde...&hl=mutagenesis

I designed a primer-linker following thses instructions but unfortunately it worked fine when I tried with GFP (I got a band which is aproximately GFP+linker size)but it didn't work really well used as a reverse for my protein (the melting T is very low: 48C). However, I obtained the whole sequence of my protein and GFP+linker and gel purified both.

When I've tried the fusion PCR, I just mixed dNTPS, MgCl2, buffer, high fidelity polymerase and both templates (100ng each) in order to get a higher band. I run the fusion PCR for 10cycles using 48C as a melting T and I didn't get the higher band and the original templates looked faint.

What am I doing wrong? Could you please give me some tips, advice, troubleshooting, anything? I don't really know what else to do. Should I do it using restriction sites? This method without restriction sites seems more appropriate to me.

Thanks in advance

-gsamsa-

Lets solve your problem.

First can you please tell me what is Tm of your primers because what happened with me was, Tm of the GFP reverse primer and forward primer of the template was varying by 5C so I had a lot of troubles fixing it (first to realise that this is 'the' issue)
In my honest opinion, you annealing temperature seems to low, I am using annealing temp of 60 or 61C at this moment for such reactions.

Your methodology otherwise seems right and yes, gel purification of PCRed GFP and your DNA of interest is very essential otherwise you end up getting same products in fusion PCR and no fused product as such because of the presence of the old primers. Watch that out.

If you are not too sure of the annealing temperature, run a gradient PCR and run 25 cycles PCR, let it run overnight !! No need of DMSO (I have never used this in my life yet)
Post if your issue still persists

I didn't understand this though

QUOTE

but it didn't work really well used as a reverse for my protein (the melting T is very low: 48C)

-Jiang M-