26 replies to this topic

[edss]

mario2004:D
Thank you very much for your help!!!


edss

[turtle]

Mario is right, the distance isn't important. I generally try to design my GSPs far enough 3' that I can also squeeze in a sequencing primer 5' of te nested GSP for walking upstream. I direct sequence my products, and find that having the sequencing primer nested inside the GSP works better than just using the nested GSP as a sequencing primer.
Turtle

[edss]

thank you for your help!
may i ask which software do you use to decide the TM values of the primers?

[without00]

It's already March... but I just wanted to briefly mention about my experience with Seegene's DNA Walking SpeedUp Kit that kiwi mentioned. I had a friend who tried it to do BAC cloning and she recommended it to me.
I had a piece of known seuqence and wanted to find out the sequence in the unknown region.
It was very easy to use and I had about 1Kb for each walking. I think the advantage of using this kit is that you only get real products (save so much time).
However, you have to design 3 target specific primers(TSPs) for nested PCR and it's quite crucial to have good TSPs for good result.

If you do try, don't forget to complete 3 PCR reactions because I didn't get any bands until the 3rd PCR.

They have a small pack.. 10 reaction.. that's what I first tried..

[Sequencer]

[i hope you could elaborate on the TSPs... I am also interested of making use of seegene... how much did it cost? thanks!

[pcrman]

[i hope you could elaborate on the TSPs... I am also interested of making use of seegene... how much did it cost? thanks!

<{POST_SNAPBACK}>

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.

[seqman]

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.

<{POST_SNAPBACK}>

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.

[Arqwen]

Universal Genome Walker is good, or else you could try TAIL-PCR (thermal assymentric interlaced PCR)
I have used both and I like both methods - although I recommend adapting the TAIL PCR cycles from what is on most papers

[ulujm]

what is genome walking?

[laoz]

I did use Seegene's DNA Walking SpeedUp Kit, because I want to find the integration site of one of my transgenic line.
I already did Southern to distinguish the one band which I thought it should include integration site, I cut the genome DNA 2-3Kb franking this band, then perform DNA walking, it turn out 3 more integration sites, definitely not right, after sequencing, I found the GT repeat in my transgenic DNA always connect to some different genomic DNA, giving false positive.
Hope this unhappy experience can help trouble shooting.
Laoz

Edited by laoz, 01 December 2009 - 08:29 AM.

[Rupam]

The GenomeWalker Human Kit contains four premade libraries of adaptor-ligated, species-specific genomic DNA fragments. These libraries are constructed from highly pure genomic DNA of high molecular weight. The DNA is digested separately with four different restriction enzymes and ligated to the specially designed GenomeWalker Adaptor. Each kit contains the reagents necessary for up to 20 walks with each library, and 150 primary and 300 nested PCR reactions
This might help.

for further queries:[url="[url]http://www.wiziq.com/tutorial/biology"]biology[/url] tutorial[/url]

Regards
Rupam

[dick123]

what brand prefer piz suggest me.