I recently posted a question concerning removal of residual Big Dye
terminators and I received many helpful answers. Thank you. All the
answers are shown below. I have found that the protocol with G-50
filled microtiter filter plates works very well. We use a Heraeus
Megafuge 1.0 and centrifuge at 2000 rcf for 3 min to pack the columns.
After that we add the sequencing product and centrifuge again at 2000
rcf for 3 min.
Best regards,
Peter Bjarke Olsen
Bioinformatics and DNA Sequencing
Novo Nordisk A/S
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Peter,
I use the old sodium acetate/95% ethanol pptn method for the big dyes
and found, with a little
tweaking, it works. I had to play around with spin times, but found
the most essential step was the ice
pptn (although you don't use this?), so it might be worth not pptning
them for so long after adding the
isoprpanol, or maybe cutting the spin times.
These pptn steps all seem to react diiferently in everybodys hands
(and different equipment too) -
play with spin times and rpm's until you get one that works, I think
it's the only way forward!
Hope this helps you some.
Regards,
Stuart.
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EdgeBiosystems(formally AGTC) have developed a protocol for cleaning
up BigDyes with their 96 plate(spin for 2min 325g and final spin for
3min 325g). We put in a wash before adding the sample just to remove
residual buffer. We only do half reactions(4ul reagent in 10ul total
vol.) I just started using the plate for cleanup but seem to work
fine? Martin
--------------------------------------------------------------- Peter,
You might want to check out protocol page on my web site.
http://www.genome.ou.edu
It has our entire protocol book on line and you can search for
sephadex or G-50 to find the protocol we use for 96 well
G-50 filled microtiter filter plates. It's cheep, easy and works.
Cheers........bruce
**********************************************************************
*** Bruce A. Roe, Ph.D George Lynn Cross Research Professor
Department of Chemistry and Biochemistry
University of Oklahoma, Norman, OK 73019-0370, U.S.A.
Phone: (405) 325-4912 or 7610; FAX: (405) 325-7762; e-mail:
broe at ou.edu ********************** http://www.genome.ou.edu/
************************
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Dear Dr. Olsen,
We use Big-dye routinly here in normal microcentrifuge tubes. We wash
the pellets with 70% ethanlo and the data are not to bad. Most of
them may have a little problem in the very begining of the sequernces
(0 to 50 maxium). some of thm are very good. I think the key is
remove all of the ethanol in both precipitation and washing stages.
We are thinking about using 96-wells to do the reactions in the very
near future. Could you give me some information about it, such as,
where do you get the plates (ABI's will be very expensive), what
centrifuge you are using, etc. Any trick to use the plates?
Any information would be very useful to me.
All of the best.
Yan Li
**************************************** Oswel DNA Sequencing E-mail:
sequencing at oswel.com
Lab 3145, Biological and Medical Scienced Building, Unibersity of
Southampton, Bassett Crescent East Southampton SO16 7PX
Tel: (01703) 594199
Fax: (01703) 594430
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Dr. Bjarke,
Let me tell you up front that I am an employee of Perkin-Elmer. Just
recently
a customer of ours told me that he found a great way to get rid of dye
blobs. He says he puts some EDTA in the sample after thermal cycling
and before the ppt. step. He didn't give me an exact protocol but said
he used up to 20mmol EDTA. I am not sure but I assume that means the
concentration in the final sample before ppt. We will be trying this
out in our labs, but it sounds easy enough to experiment with, and may
help.
Regards
Dave Clark
David Clark
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This is the method that we use for removing unincorporated
terminators. It adds additional steps to the precipitation method but
the advantage is that you remove all the garbage with this method and
you start reading sequence at base #1 and the tracking of the gel is
usually better. We use Millipore Multiscreen filtration plates filled
with G50 resin for this method. You run your samples through the G50
resin and collect in another 96 well plate and then precipitate as
usual. You can ask Millipore for this document: Lit No. TN 053. Title:
Dye Terminator Removal Using Multiscreen 96 well Filtration Plates.
The supplies you need for this are:
MAVHN4550 Multiscreen HV plates 50/pkg $483.00 (MAVHN4510 would
be for 10/pkg)
(We wash out these plates and reuse them. It works great!)
MACL09645 Multiscreen 45 ul column loader $195
(this is a cool device used to load dry sephadex G50 to each well of
a plate)
S2ER088V7 Multiscreen centrifuge alignment frame $15
(we don't use this device with our centrifuge, but you might need it
to keep your collection plate in line with the filtration plate.)
Sephadex G50 Pharmacia 17-0573-02 (100g) $182
This method is very cost effective if you reuse the plates. They are
easy to clean out. We just use dH2O.
I hope this helps,
Joe Cook
Core Sequencing Facility
Bristol-Myers Squibb
------------------------------------------------------------ Saw you
inquiry on the AUTOMATED DNA SEQUENCING NEWSGROUP.
The dye blobs are due to the co-precipitation of dye-ddNTPs with the
labeled DNA (this is fairly common knowledge). One method WE HAVE
FOUND HERE AT NEN to make the residual dye-ddNTPs less likely to
precipitate when ethanol precipitating is to cleave off the phosphates
from the dye-ddNTPs BEFORE adding ethanol.
Here's a procedure you might try:
Dilute CIP (calf intestinal alk. phosphatase) to 1 U/uL in 50 mM
Tris-HCl, pH 7.6, 2 mM MgCl2.
Following the sequencing reaction, add 1 uL CIP ( 1 U) per sequencing
reaction.
Incubate @37 dC for 20 min. (longer OK)
Ethanol precipitate as usual, or try method below if different:
Add 35 uL of chilled 95% EthOH to a 10 uL aliquot of reaction.
Incubate on ice for 10 min.
Centrifuge at 13,000 rpm for 20 min. Pipet off EthOH and discard. Wash
pellet with ~75 uL chilled 70% EthOH (DO NOT resuspend pellet). Pipet
off EthOH wash and discard.
Dry Pellet.
Dissolve in loading buffer and electrophoresis.
THE PRIMARY SOURCE for Dye Labeled NUCLEOTIDES,
Philip R. Buzby, Ph.D.
Nonrad. Nucleotide R&D Phone: 617-350-9343
NEN Life Science Products, Inc. Voice Mail: 800-446-0035, 1,
9343
PO BOX 199151 FAX: 617-350-9658
Boston, MA 02119 E-Mail: buzbypr at nenlifesci.com
