Dear Evoldir members, Here are the replies I received to my original posting: "I'd be grateful to anyone who could tell me about their experiences of diluting BigDye for the ABI 310 capillary sequencer. I'd also be interested to hear from anyone who has tried the dLUTE SEQ DNA Sequencing BigDye Dilution Reagent on the ABI 3130." For those who asked, information about the dLUTE SEQ DNA sequencing reagent can be found at http://www.nucleics.com/DNA_sequencing_tools/DNA-sequencing-dlute-seq.html Thanks, Paul --- Dear Dr. Bloor, While I can't address dilluting bigdye, I can say that I've used what we call `quarter reactions.` This saves us considerable reagents. Taken from my institute's Nucleic Acid facility: "1/4 Reaction: For each reaction add the following reagents to individual tubes: Terminator Ready Reaction mix (Big Dye) 2 ul Big Dye Sequencing Buffer 5X 1 ul Primer 1ul (3.2 pmol) Template See table for Template guidelines Water To 11 ul Total volume 11 ul" http://mercury.bio.uaf.edu/core/protocols/_cyclesequencingrxn.html This has worked very well for us, and I reliably get clear sequences. I hope this saves you as much time and money as it has saved us. Best Wishes, Kevin Colson --- Hi Paul,       You can dilute the big dye to 1/8 reactions routinely (compared to ABI protocols) and sometimes you can get away with 1/16, but it also depends on amount of template and length of your PCR product. We did this with a 310, then 3100, and now 3130.  It is very routinely done.       I haven't tried the reagent that you mention below and I am interested to find out what you hear. Who makes that reagent?       thanks, Sarah --- BigDye v3.1 my sequencing reactions are 1ul BigDye 1ul 3.33uM primer 1ul PCR product (EXO-SAP clean) 7ul 1x BigDye buffer --- Hi Paul, I have used up to 1/8 dilution of Big Dye terminators on various ABi platforms. Most commonly I use 0.5 ul in 10 ul reaction. Never had a problem with diluted Big Dye. ABi want us to use more because it's so damn expensive! Some people also use home-made buffer rather than shelling out for their original stock. Of course ABi claim the reactions don't work as well but big facilities make their own on a regular basis. Good luck, Birgit --- Hi, I use 1 to 0.3 uL big dye per sequencing reaction (10uL). Maybe one can go less. I have tried the dLUTE kit with no success. I only tried it twice but neither time did I get any results and I gave up. Because it that kit differs at every step (has its own precipitation, for example) troubleshooting will require commitment. Maybe it is worth it. Good luck, Kathryn --- I always use a 1/8 dilution (0.5 ul Ready Reaction mix in a 10 ul reaction, run on a 3130xl sequencer if that makes a difference), with good results.  I don't think going above 1/4 improves things much (though I admit I haven't done many controlled experiments).  There are people who use 1/16 or less, but the few times I've tried it, I've had poor results.  I think your PCR has to be extremely clean, and the amounts of primer and template DNA exactly right, for that to work. The full recipe I use per reaction is 0.5 ul BigDye, 1.8 ul 5x buffer, 2.4 ul water, 0.3 ul primer (10 uM), 5 ul template+water (ratio depending on template concentration). Karl --- Dear Paul, In my previous lab, we successfully used 0.5 ul BD/10 ul reaction. I've attached my excel template that calculates the master mix with the appropriate amount of dilution buffer (2nd sheet also has the template amounts we used). Given the ridiculously high price of the dilution buffer from ABI, I asked for recipes on evoldir a few years back and have attached the answers I got. We ended up purchasing the buffer from Teknova, as recommended by Rosanna Giordano (see product numbers in the .doc file) you get a huge amount for ~$40 and it worked great. Good luck! Leslie --- I have no experience with the 310, but with the 37030xl. But i can say that 0.5 ul in a 10ul reactionm works just fine. Our reactions contain 5.9 ul ddH2O, 2 ul 2x seq buffer (we make our own), 0.6 ul primer (10 nmol), 0.5 ul BigDye, 1 ul PCR (SAP/EXO cleaned and undiluted). This works in 95% of situations. hope this helps --mark ---- I've gotten good results (just as good as at full strength) using 1/4X and 1/8X dilutions.  I got the idea from Travis Glenn (who probably has recipes at his website).  I use a 310. --- We did some tests and used to dilute ours to ¼ what is suggested by ABI, and that worked fine. I'd be curious to hear what other people have found. Camille --- Dear Paul, >From my perspective, the 310 should not be very different from the other capillary sequencers. However, I used it very long time ago. The BigDye dilution will depend mainly on the sequencing cleanup protocol, as well as type of template. With Sephadex I won't recommend to go below 1/16. Please find attached BigDye calculator table that we use in our facility along with our sequencing and PCR  protocols. If you plan to use Agencourt CleanSEQ be prepared that you'll have to optimize template concentration in the sequencing reaction in order to get optimal results. Another important factor for signal strength is an injection solution. Formamide gives lower signal, but stabilizes sequencing reaction for longer time, compare to EDTA. For a start, try 1/8th dilution - this is the safe dilution which is working well both with plasmids and PCR products. For PCR products follow ABI guidelines  for  template  concentrations (they are a good start point for template optimization for diluted BigDye as well). This concentration is also working well with ethanol precipitation cleanup protocols. We don't use any commercial buffers for dilution (the recipe is provided in sequencing pdf). You will notice that we use trehalose both in PCR and sequencing - I highly recommend it! You will also notice that we don't clean up our PCR products, but we use very low concentrations of dNTP and primers in PCR (see attached document). Hope this information is helpful. Let me know if you have any questions. Best regards, Nataly --- Hi Paul. The sequencing CORE at University of Michigan does 1/8 the recommended volume amount. I copied/pasted their response below.  I would be interested to hear about any other replies to your query.  I too am trying to find ways to reduce costs associated with running a 3130xl.  Cheers - Jeff Johnson. +++++ We use 1/8th as much BigDye as ABI recommends, and the reaction volume is much lower than their standard conditions. This has two effects: it cuts cost dramatically, and it causes marginal samples to fail. We are hard-nosed about the concentration of samples, because we know that our low-cost recipes are fairly unforgiving of template quantity. It is possible to cut the dye concentrations further, but only if your samples are very good quality, and very consistent. Genome centers can do this, because *they* make all the samples, *they* ensure that they are consistent, and *they* don't care if 10% of them fail. Cores can't get away with that  tactic - our clients would howl if there was a 10% failure rate on samples that would have worked with a bit more dye! -- Paul Bloor Coordinador/Investigador Principal Unidad de Especies Silvestres Instituto de Genética Universidad Nacional de Colombia Bogotá D.C - COLOMBIA Tel.: +57-1-3165-000 Ext. 11611 Fax.: +57-1-316-5526 alt. email: pbloor@unal.edu.co pbloor@gmail.com