HI Amit
It is a method to isolate the sequences upstream of a known sequence.This is usually done to isolate promoters or flanking sequences of a gene. Refer Clontech's Genome Walker kit' user Manual

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I have used the seegen' s kit in fungal experiments, it is easy to use, and well performed.

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Hi

Is there any company other then clontech which offer genome walking kit or related technique to find out unknown sequences from DNA.?

regards

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Hi everyone,
I have some problems with primer design in my Genome Walker experiments. I decided to use Universal Genome Walker Kit (CLONTECH). The problem is with Tm of primers. As sb said earlier different programs give you diffrent Tm. Can anyone tell me what is the best method used by program to assume Tm? Do I have to take into account NN, basic or salt adjusted (nearest neighbour, 2AT+4GC and %GC respectively)?
Thanks very much for help:)

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hi ,everyone !
i have used the Universal BD Genome walker kit from CLONTECHfor genome walking in plants. But it didn't work very well. i always got a band of 5~6kb, however when cloned into pmd-t vector, it was only 2kb.

I don't know what is wrong and how to deal with it ? Can any one help me ?

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I just wanted to say that I am experiencing the same problem. I am working with plant DNA as well (specifically potato) and I have isolated a ~2kb band and gel purified it and then cloned the purified product into pGEM-T easy vector and when I did PCR on the subsequent colonies it indicated that I only had ~400bp.
Although, I am not able to help in this situation I wanted you to know that you are not the only one experiencing it. If you figure it out let me know and I will do the same.

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I also have been running into problems with the Universal genome walker kit. I am trying to clone a promoter from Chinese chestnut and had to design my primers from a partial known sequence.

My first walk produced bands at 3 kb, ~1750kb, and smaller sized fragments. Upon cloning (TA cloning kit), however, only the smaller fragments were successful. At first the PCR protocol was not successful, - - my GSP1 had a Tm of 71 & GSP2 had a Tm of 70. I had to redesign my GSP1 with a new Tm of 61 and then use a 3-step pcr program.

I am now conducting a second walk in both directions with new GSP's. Is it necessary to conduct both the primary and secondary pcr again, or should I try a pcr with my new GSP & AP2?

Does anyone have optimizations for using a mj research ptc-150 minicycler?

Thanks
Biotech Stumpie

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I am doing genome walking to fish the promoter sequences of my gene by using PCR LA kit (TaKaRa). I performed restriction cuttiing with six enzymes and ligated with the casette provided by the kit. When I did PCR with my specific primer of known region and casette primer. It had only short PCR product 400 bp obtained from Sau3AI cut chromsomal fragments. Can anyone give me the suggestion why I obtained only short PCR fragment and how can I solved the problem to get the long PCR fragment. By using this kit how can I decided with restiction enzyme to be chosen to cut the chromosome before doing genome walking. The kit has EcoRI, HindIII, PstI, SalI, Sau3AI and XbaI cassettes.
Thank you

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I am trying to walk a 9 Kb gene, but so far I have no success in getting fragment. I always get 300 to 500bp fragment. Is it my primers doing this or is there any other way that ican obtain a bigger frament.

please help

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If your gene is cloned and is only 9Kb, then you can sequence it directly without attempting to subclone it. Sequence in from both ends as far as possible. Choose two primers near the end of reliable sequence from those read, and sequence forward again. At the same time, choose two reverse primers from the very reliable middle of the sequence read and sequence those for verification. You can advance about 800 bp at each end per round, or about 1600 bp, so in about six rounds you will have the complete sequence. You can speed this up even more if you know of a starting sequence in the middle, of course. This is all far easier and more straightforward than the complex "kit" protocols of chewing back ends, partial cutting with enzymes, subcloning, transposon insertion, whatever.

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