G + C rich sequences
nishir at ohsu.edu
nishir at ohsu.edu
Fri Jul 30 11:47:32 EST 1993
In article <2C5933B7 at coursmtp.nrc.ca> Baldauf at imb.lan.nrc.ca writes:
>>formamide in 5xSSC + 5x Denhardts + 1% SDS + 10% dextran sulphate +
>>250micrograms/ml of salmon sperm DNA at 42 degrees; we wash with 0.1X SSC +
>You might want to try decreasing the dextran sulphate, it's notorious for
>giving background. Use 5% or even leave it out altogether. Unless you're
>looking for something very rare in a large complex genome, or using a poorly
>matched probe, you probably don't need it.
Actually, we are trying to look for something rare and related (not matched) to
the sequence of our probe. The dextran sulphate did not give us high
background when we did the same (soln and hyb and wash conditions with a
different probe (that had a more sensible G + C content)) on the same genomic
blot. I made a mistake about the wash conditions...since we are looking for
something related, we are using washes at 2x SSC at 50 deg.
A whole slew of thoughts/questions has come up...
Another idea came in to try slightly higher hybridization temps (perhaps to
reduce secondary structure of the probe)- what's the experience with this--
has anyone tried >55 deg without formamide or >42 deg with 50% formamide with
DNA probes? The problem here is to still allow hybridization to "related"
sequences but to discourage self- binding of the probe. I tried looking at a
paper on hybridization kinetics and it totally baffled me. If someone could
explain in plain English how stringency varies with temp, I would appreciate it
(eg., at "high" stringency how long must the length of match be to withstand
denaturing; for each drop of 5 deg in temp, how much "mismatch" in this stretch
be allowed? What if the sequence identity is not a string of base matches but
is a base match consistently every third residue.. will such sequences not
every be picked up even at lowered stringency?)
And finally, what does formamide do? A suggestion was made that it complicates
an already complex equation, so why not leave it out. I know that formamide
destabilizes DNA hybridization, so if formamide is included, you can hyb at
lower temps than without formamide. So many of us just use hyb solns that
other people have used. I want to understand and get a better sense of the
variables. Does formamide affect background? Does it make it easier to vary
stringency by varying temperature? Or is it just a pain in the you-know-what?
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