Primer walking - (Nov/07/2006 )
Can anyone tell me how to use primer walking to sequence a 10 kb full length?
You sequence the insert from both ends (assuming you have vector primers known at both ends). About 600-800 bp into the sequence you get, choose a new primer, synthesize it, and sequence again. If you want to be careful, sequence backward as well. Since you get 1500 bp or so (from both ends) each time, this will take about six or seven repeats. Assemble the sequences with (for example) Vector NTI.
Thanks, phage434! Besides Vector NTI, are there any good and free softwares to assemble sequences?