Reply #1
I talked to the GenPak people and they have a new extender
called Half-BD suppored to be formulated for the BigDyes. They
claim to have some substance in there that keeps things stable.
Don't know if its true may in fact just be buffer. We have used
the regular half-term with BigDyes for a couple of months now with
very good results. IF people send us good template. Getting more
rare all the time.
Reply #2
I have consulted with our local ABI technical consultant Neil Kennedy
regarding the use of halfterm. And here is what he told me:
"Half-Term does not help your sequencing reaction, it only provides what
our
buffer does at a higher cost. So, if you plan to do reduced volume
reactions
you can either use our buffer or half-term. You have the choice of two
buffers."
Reply #3 (from our Field Application Specialist Nathalie Bernard)
Hello Dr Pon,
Someone "intercepted" your message on the bulletin board
>does anyone know if it is possible to purchase sequencing buffer for FS
or
>dRhod kits separately?
Yes, the buffer available is the 5X buffer that we provide (Part Number
361028C,
price $25/ml, to be use at 2.5X concentration, I think this is cheaper
than the
Halfterm product...) Note: minimum order of 5 ml, price in $US
>Will the Big-Dye buffer from ABI serve the same purpose
>when used with FS or dRhod kits?
Yes, no problem
Reply #4 (again from Nathalie Bernard)
>I assume that the 5X buffer is meant to be used in a 1:1 ratio
>with the sequencing kit mix, just like the half-term. However, why do
they
>call it 5X buffer (and not 2x)?
In fact, no, it is really 5X buffer, from the core sequencing kits ; it
was
meant to be used in those kits at 4 microliters in a 20 microliters
total
reaction volume.
The ready reaction mix is in fact at a concentration of 2.5X (since you
add 8
microliters to a 20 microliters reaction). When you want to use the
dilution
buffer, you need to dilute it first at 2.5x (1:1, with water) then
instead of
using 8 microliters of ready reaction BDT mix, you need to put in your
tube, for
a 20 microliters final volume :
- 4 microliters of RR mix and 4 microliters of 2.5X buffer, if you want
to
dilute it 2 times
- 2 microliters of RR mix and 6 microliters of 2.5X buffer, if you want
to
dilute it 4 times
- 1 microliter of RR mix and 7 microliters of 2.5X buffer, if you want
to dilute
it 8 times
(I have a customer in Winnipeg diluting the RR mix up to 16 times ! ;
you need
of course good template to do this, so I give you the info but I don't
certify
it will work with all your customers' DNA ;-) !)
I hope the information is a little bit more clear now. If you have
questions,
don't hesitate to contact me
>I will also forward your information to the rest of the ABRF
>e-mail list for everyones information, if you don't mind.
Please don't hesitate ! This is indeed good info for everyone !
Reply #5, from Hal Hills, Iowa State (copy of letter to his sequencing
users)
To: To those who want to do their own DNA sequencing reactions.
From: DSSF, Hal Hills
Subject: Big Dye dilutions
This is information I received from PE about diluting the big dyes. I
have done the dilution series and got acceptable results using 1 ul
premix and 7 ul dilution buffer in a 20 ul reaction. It is necessary to
adjust template and primer amounts to get the best results with a 1:7
dilution. The facility has not been able to make the dilution as much
as we would like because of the varied amounts of DNA sent by clients.
We are still trying to find acceptable levels of dilution which will
work for all clients. It is best that you stay with a 20 ul reaction.
A column cleanup is preferable to precipitation. We are finding that
some users attempting to precipitate BigDyes are having serious carry
over in the precipitated samples and they are causing problems in
adjoining lanes on the gel. To save the cost of columns, you can follow
one of the precipitation procedures supplied with the kit. However, one
of our users found that ammonium acetate precipitation worked better and
did not cause dye blobs which interfere with adjoing lanes. It also had
lower salt content in the dried down reaction products. The facility
is continuing to use spin columns from Princeton Separations.
The 100 reaction big dye kit is catalog # 4303149. You should be able
to do 300-400 rxns with the kit or more if you are working with short
PCR products. Bruce Roe at Oklahoma is pushing the dilution much
further.
The info in quotes is from PE.
=93Hope this helps, I would not push the reaction to 1/8 but 1/2, 3/8, or
1/4 is the most I would push things at this time. Let me know what you
find out and any other opinions you have.
Ready reaction mix contains: 200 mM Tris, 5 mM MgCl2
This is equivalent to a 2.5x buffer concentration . Normally 8 uL
of RR mix is used in a 20 uL reaction. Therefore, the final
concentration (ie. 1x concentration) of buffer components is: 80 mM
Tris, 2 mM MgCl2 at pH 9.0
5 x sequencing buffer (in core kits) contains: 400 mM Tris 10
mM MgCl2 at pH 9.0
Therefore, the easiest way of diluting is to use 2.5 x
concentration sequencing buffer and add sufficient to bring the RR
premix + buffer total to make 8 uL. For example in a 20 uL total
reaction volume:
4 ul RR premix + 4 uL 2.5x buffer in a total 20 uL rxtn *
3 uL RR premix + 5 uL 2.5x buffer in a total 20 uL rxtn *
2 uL RR premix + 6 uL 2.5x buffer in a total 20 uL rxtn *
1 uL RR premix + 7 uL 2.5x buffer in a total 20 uL rxtn
This keeps the final concentration of Tris and MgCl2 as for a full
volume reaction (20 uL) with 8 uL RR premix.=94
Conclusions
According to PE/ABD we should be able to use their buffer in place
of the more expensive Half-term buffer. The only problem is that this
buffer is presently available only as a special custom packaging item.
One could also try making their own 400 mM Tris, 10 mM MgCl2, pH 9.0
buffer. I have ordered some of the PE/ABD buffer and will try it out in
place of the Half-term buffer as soon as it comes in. Even at $25/ml, it
will be a big savings for us. I just hope that when ABI realizes the
usefulness of this item, they won't jack up the price.
Best Regards
Richard T. Pon
University Core DNA Services
University of Calgary
tel: 403-220-4277
fax: 403-283-4907
e-mail: rtpon@acs.ucalgary.ca, dnalab@ac.ucalgary.ca