Rocky Ho <RockyHo at cuhk.edu.hk> wrote in message
news:7lnv4e$h0t at net.bio.net...
> I am now using 8 µl BigDye terminator reagent mix for 20 µl sequencing
> reaction. Is there any possible suggestion for reducing the use of the
> reagent?
>> - Rocky Ho
Hi,
yes we can
The big dye Dilution buffer (Dbuf) is 200mM Tris pH=9.0 plus 5mM MgCl2. This
buffer has the same concentration Tris and MgCl2 as the big dye Ready
Reaction mix (RRmix). The idea , ofcourse , is that you can now mix the two
in any ratio without changing the buffer components and save money.
See also the bigdye manual file
http://www2.perkin-elmer.com/ab/techsupp/310.html for remarks on template
length (sequencing Bac templates)
Standard is :
1) 8.0 ul RRmix
2) Template a) single stranded DNA 50 - 100 ng
OR b) double stranded DNA 200 - 500 ng
OR c) PCR product DNA 30 - 90 ng
3) 3.2 pmol sequencing primer
4) Add water to adjust the reaction volume to 20ul.
If you want to save on the expensive RRmix ( fl 30.0 / reaction) than you
can try to dilute it. In the perkin elmer terminology , replacing the 8.0ul
RRmix in 1) with :
4.0 ul Dbuf + 4.0 ul RRmix gives a 1/2 reaction
6.0 ul Dbuf + 2.0 ul RRmix gives a 1/4 reaction
7.0 ul Dbuf + 1.0 ul RRmix gives a 1/8 reaction
(components 2) , 3) and 4) can then be kept constant.
On page 18 is a guideline for sequencing BAC DNA which is , of course , a
longer template .
1) 8.0 - 16ul RRmix
2) BAC DNA template 300 -600 ng
3) 4 - 8 pmol sequencing primer
4) Add water to adjust the reaction volume to 20 - 40ul
As you can see from this : the longer the template the more RRmix is needed.
This makes sense to me. If the template is longer more dNTP and ddNTP is
needed for the longer extension products. Also true even if you can not
detect these longer extension products because the resolution of the machine
is not better. So in my opnion ( and we did some experiments to back this
up) : if you sequence 500bp from a 600bp template than you will need less
RRmix than if you sequence the same 500bp from a 6000bp template. The
discussion was about the effect of dilution on the detected sequence. At
first we were very surprised that it was possible to do a 1/2 or 1/4 or even
a 1/16 reaction ; nice clear results with not one N called. However when we
tried to do this in a more systemic way with a well defined vector sequence
we quickly noticed the called A , C , T and G's were just wrong. So a
sequence from the ABI machine with no N's is not the same as an error free
sequence!!!!!!!!!!!! The specifications of perkin elmer : >98.5% correct for
base 20 till base 420 (for the short capillary) is easily met if you do
their protocoll. In our hands the dilution only works for the shorter
templates. ( As is in their protocoll!!!)The error rate plotted against the
template length is some sort of power function. So if you do a 1/8 reaction
on a template of 3kbp than there will be much more than 1.5% error between
base 20 and 420 ; however most of the errors will still be after approx.
base 200. So if you just need the first 50 - 100 of those 3kbp than dilution
is OK. To be practical we determined the error rate for a (known model )
template of 2kbp. A 1/2 reaction was just within the specifications ( < 1.5%
error from base 20 - base 420). We now routinely do 1/2 reactions but never
on template longer than 1.5kbp (form agarose gels)
Good luck
Gys
