Posted 18 September 2004 - 07:05 PM
Thank you very much for your help!!!
Posted 20 September 2004 - 08:40 AM
Posted 29 September 2004 - 12:11 AM
may i ask which software do you use to decide the TM values of the primers?
Posted 04 March 2005 - 06:35 PM
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..
Posted 21 March 2005 - 10:47 PM
Posted 22 March 2005 - 05:41 PM
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.
Posted 25 March 2005 - 12:05 PM
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.
Posted 06 August 2009 - 02:36 AM
I have used both and I like both methods - although I recommend adapting the TAIL PCR cycles from what is on most papers
Posted 01 December 2009 - 08:28 AM
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.
Edited by laoz, 01 December 2009 - 08:29 AM.
Posted 30 August 2010 - 12:43 PM
This might help.
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