Dye blobs-follow up

PBO PBO at novo.dk
Thu Aug 20 16:40:08 EST 1998


     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
     
     --------------------------------------------------------------------- 
     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.
     -------------------------------------------------------------------- 
     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/ 
     ************************
     ---------------------------------------------------------------------- 
     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
     --------------------------------------------------------------------- 
     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
     -------------------------------------------------------------------- 
     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




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