TSPs are just primers you will use to amplify unknow DNA sequence with a link primer in genome walking. You can design it by yourself and order it from any company.

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i aslo sent this messeage to without00.

i am using seegene kit, but i've got nonspecific sequence. it is amplified by ACP primers. so i am wondering how too design an effective TSP primer.

according to the mannual, ACP primer-binding sites(5-XGGTC-3) upstream of TSP1 or in/between TSP primers should be avoided. why is this a big deal? there must be a limit of distance for the distance. i mean we should only consider a certain distance upstream, like within 1kb. am i right?

how about those binding sites downstream of the TSP primers? doesn't matter?

how long fragment can you get using this kit? can we use long PCR system instead.

hope you can share your expereince with us.

thanks a lot.

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Hi all,
I have recently used Clonetech's Universal genome walker kit with pretty good results.  I did have to do a bit of tweeking though. 
First of all, the annealing Temps. suggested in the kit didn't work, not on the controls or my specific samples.  I went to gradient PCR to figure out the best temp for each combination of primers (adapter & specific).  Next, after doing all of this trouble-shooting, I was out of Taq and didn't want to spend an enormous amount of money to replace their proprietary Taq, so I tested a few and found that Invitrogen's Platinum Hi Fidelity Taq worked well at about 1/6 the cost of their Taq. 
As for cloning vs. direct sequencing, I never direct sequence unless I'm > 90% sure I'm only amplifying one product.  When walking through a 'library', there is a high chance that you will get more than one product in any PCR reaction or gel purified band for that matter.  I use pGEM-T easy, love it, have cloned larger inserts (2.5 kb from this walk) and it's easy to sequence using standard primers.
My organism is ryegrass and I've obtained additional 5' and 3' sequences, but still not to the ends of the gene.  The trouble is that there are introns in my genomic DNA, so now I'll resort to RACE!

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hello segman,

i am sorry for this late reply.
I think the theory behind Seegene's DNA Walking kit is pretty complicated... from what I understand, TSP1 is crucial. They employed ACP technology in this kit, which is supposed to maximize PCR specificity. Therefore, as long as the products from the first PCR is specific, you will continue to get specific products in the following PCR reactions(nested PCR reactions).

if you have got non-specific bands, i think you should write to seegene. they have some sort of back-up sheet for technical support... my friend needed some technical support for DNA Walking kit and she resolved her problem that way.
She was told that the non-specific bands from ACP primers was created because there was no target product created during the first PCR reaction. She, then, had to change her TSP 1 in the second trial. The job of ACP primer in this kit is to randomly bind to any XGGTC site in your unknown region. That's why you should avoid this sequence between TSPs.

I got a squence upto about 1.3 Kb. It really depends on the chance of XGGTC appearance in your template.. I mean, mathmatically XGGTC should exist in every 1Kb, but it depends on your template.

I also heard that ACP doesn't work with enzymes that has a proofreading activity. This enzyme is supposed to interfere the activity of DW-ACP primer.


oh, by the way, this is for sequencer...
TSP is target specific sequences. You have to design 3 TSPs in the region with known sequences for nested primer.
10 reaction kit was about USD240. i am not sure what it costs outside of US though.. you can download the manuals from their website. they mention criteria for TSP design in the manual... hope this helps!

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Hi

Just for your information,
i have used Seegene's DNA Walking SpeedUp Kit before. erm... with the ACP primer technology in the kit, we managed to get the promoter region of the gene.

After the third round PCR, we sent the amplicon for direct sequencing without cloning. The result was ok.

good luck and all the best...

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How does this approach compare to inverse-PCR? It seems to me that inverse PCR is more straightforward, and requires only standard reagents, a little cleverness, and far less optimization.

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Hi there
I have been using TOPO TA cloning kit routinely for cloning and then sequencing. i have always felt confortable with M13 preimers for sequencing. If your product size is really big i mean more than 4-5k then go for TOPO XL. you can use XL up 35-40K with great efficiency.
Good luck

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can you tell me what is genome walking.........
regards
amit

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Hi

Have a look at this

Devic et al., Plant Physiol. Biochem, 1997, 35 (4)

I have been in this lab' to learn the technic but I have never used Clontech's kit. I find it's a waste of money.

You can just make the AP1, AP2, and GSPs and buy the whole lot separately. Saves you some cash.

It always worked fine in my hands but it is important to have at least 5 different "libraries" because you don't always have results with all of them.

The use of a gradient thermocycler seems to bee a good idea too..

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I have used the Universal BD Genome walker kit from CLONTECHfor genome walking in plants.It worked well but got only a700 bp fragment upstream

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