Genome walking - (Sep/02/2004 )
Sequencer on Mar 22 2005, 12:47 AM said:
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.
pcrman on Mar 22 2005, 07:41 PM said:
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.
Hello everyone. Nice to meet you all.
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
what is genome walking?
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
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:biology tutorial
Regards
Rupam