Genome walking - (Sep/02/2004 )
QUOTE (phage434 @ Aug 10 2007, 07:48 AM)
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.
I am sorry my question was not quite clear This 9kb gene is the gene of Arabidopsis and I am trying to get the same gene in Eucalyptus sp. So I am using the genome walking procedure to get the same gene in my species. So when I use the genome walking kit I only get 300 bp fragment. I am using 8 different libraries. But no success in getting larger fragments so far. Please help
-juliegeorge-
QUOTE (Indian @ May 14 2006, 01:11 AM)
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
Actually it can go upto 1kb..
-bhappy-