Thanks for all of the suggestions, here are the replies that I have
TAIL PCR is very easy and, in my hands, has been very successful.
Dear Dr Fineberg:
I ever isolated a water channel-like gene from Methanobacterium
Marburg genome with single primer method.I designed several short
forward and reverse
primers(14-18bp) corresponding to the known sequence and only one primer
was used to
amplify the target gene.After fractionation on 1.5% agarose gel,each
fragment was extracted
and was respectively uased as template for nested PCR.The fragment from
which an expected
size of nested PCR band could be apmlified was cloned and sequenced. I
used this methods to
get not only full sequence of the target gene but also 5kb 5^ upstream
and 2kb 3^ downstream
sequences within one week.The all precedure consisted of a set of
conventional PCR.I would
publish this method.I attach the manuscript for your reference.But this
method maybe only
work for small size genome
Xiaodong Ding, PhD
For some years we have been using plasmid
rescue to generate border flank sequences. We use a
T-DNA with pBR322 inside the left border. In the
T-DNA there is a single ECOR1 site to the right of the
pBR322 sequence. The protocol is.
1 Make genomic DNA
2 Make a Southern blot with ECOR1 and probe with
pBR322. This allows you to estimate the size and number
of expected recovery products.
3 Cut 5 ug of the genomic DNA with ECOR1. Clean it
up with phenol, chloroform and ethanol precipitation.
4 Ligate with T4 ligase in 1 mL of buffer overnight.
5 Ethanol precipitate and resuspend in 20 ul of T10.
6 Electroporate 1-5 uL into electrocompetent Sure cells
(Stratagene) and plate on carbenicilin (50ug/mL)
A successful experiment gives 50 to 200 colonies.
7 Grow up and mini prep colonies for plasmid DNA.
These can be cut and sized and ultimately sequenced
using a left border primer.
The method works well but takes time. The advantage of
the cloning step is that multiple left borders can be
sequenced from the same line.
Good luck with your work Ian Furner