Hi all, Thank you for the many helpful suggestions for maximizing the efficiency of our ABI 3130 polymer (POP7) use. Below you can see my original question along with all the replies I received. Our department has recently purchased an ABI 3130xl for DNA sequencing and fragment analysis. We expect that our usage of the instrument will vary, and there may be some weeks where it is only needed for a few runs. Given that, we would like to minimize our costs for consumable reagents, particularly polymer. ABI suggests changing any polymer bottle that has been on the instrument more than a week. Has anyone tried running the instrument with older polymer? If so, how old? Any adverse effects on the data or the instrument? Also, any other tips for saving on reagents during periods of inconsistent use would be greatly appreciated. Thank you, Dan Sloan Graduate Student Biology Department University of Virginia dbs4a@virginia.edu We also are running a 3130xl and I've found that the polymer works fine for 10 days. After that, the peaks get wide and more difficult to read. We only fill the polymer bottle up to how much we think we're going to use in 10 days (but that's difficult to determine due to unpredictable use). Good luck, Caren Goldberg University of Idaho ------- We have had similar issues concerning the varying usage level of ABI. We have tried the following 2 approaches of which the latter we are now applying. At first we kept the full polymer bottle that was in use in a fridge and had water bottle attached inside the machine. Then replaced the water bottle with polymer bottle when starting run. This however resulted in considerably faster electrophoresis runtime and subsequently the results were bad. Bad meaning that as normal runtime for GS 500LIZ to travel throuhg capillaries would be around 6000 datapoints the runtimes varied from 2500 to 5500 datapoints. This in turn results inconsistent in respect to fragment lengths between runs! The reason probably is that polymer became diluted as water was let to system. Now we are doing the following which seems to work fine. We have the 'full' polymer bottle in fridge and 'empty' polymer bottle attached to machine. 'Empty' polymer bottle always contains a minimum amount of polymer enough to have the tip of tube to be embedded in polymer thus preventing airbubbles to appear in system. Then depending on how many runs needs to be done and whether bubble removes etc. needs to be done we add the appropriate amount of polymer by pipetting with pipette with truncated tip. There is always some 'old' polymer in the bottle but as long as you run few runs per week this polymer should always change and should not result in elongated runtimes. If polymer gets old then you will observe elongated runtimes as but as long as GS500LIZ travels through between 5500-6500 datapoints, the results are very consistent and comparable. I hope this helps. Juha-Pekka Vähä ------- I'm using Abi310 almost on my own and many times have similar problem. I've checked that you can keep the polymer for a little longer, the longest time i run samples on the same polymer was 2 weeks and the results were still fine. I also keep the buffer for longer, usually about 10 days, but the guy from Applera told me, that it's better to be rather strict with changing buffer. Could you please post me the answers you will get, or post them on evoldir? Maybe there will be some other interesting tips. Ola Gondek Institute of Nature Conservation Polish Academy of Sciences ------- If you keep the room where the sequencer is palced airconditionned so that the temperature is below 20 degreeC, and if you just need fast short runs for sequencing small fragments (says, 300bp), the polymer remains fairly good for several weeks. But, this is offset by a shortened life of the capillaries (due to old polymer) to our experience. Good luck. De-Xing Zhang ------- POP7 lasts at least 15 days on the machine with no ill effect on band resolution or the capillary (1000+ runs). When our 3730xl and 3130xl have low usage we 1) do not put on a full bottle (recycle the old empty bottles) and 2) we do a blank water run every second or third day to keep the polymer from getting "stale" in the capillaries. Lawrence C Shimmin University of Texas Houston Health Science Center Human Genetics Center ------- We have used polymer that was slightly past expiration with good results. However, every situation is different. Nonetheless, I would take ABI's "no more than a week" with a grain of salt. Alan W. Meerow, Ph.D., Research Geneticist and Systematist USDA-ARS-SHRS, National Germplasm Repository ------- We're using a 3100 which is nearly the same as your machine. We have left polymer on the machine for more than a week (up to 2 weeks) with no ill effects. The other thing we do to cut costs is extend the length of the capillary array. ABI contends they should last 100 runs, but we've had arrays last for ~550 runs, which drastically reduces the cost per run. Good luck! Matt Carling ------- We are in a similar situation with our instrument (a 3100). After polymer reaches a week to 10 days, data really does get poor (you will see an elevated/inconsistent baseline in the raw data view that does affect your results). I have found, unfortunately, that the less-frequently the instrument is run, the more problems arise. We try to anticipate use and add only enough polymer for the number of sequences or FA samples for the week, and keep the rest at 4 degrees. A few times when I knew that I had overcalculated, I expelled the polymer into our secondary polymer container for storage in the fridge, cleaned the instrument, and left it dry for a period of non-use (you will have to remove the capillaries and store in water/buffer). This polymer seemed to work fine when I used it later. I'm not sure if that strategy would immediately translate to the 3130 (which has a polymer pump that we don't have). Perhaps you could also keep some polymer in reserve rather than starting with all 7ml on the instrument. We have considered declaring every other week "off". Although this would increase turnaround for our in-department customers, it could let them plan ahead and allow us to save on wasted consumables. Good luck, -Genevieve Croft Lab Manager, Hamilton Lab Georgetown University ------- A PI I work with swears by installing an air conditioner over the ABI as the cooling greatly increases the life of the polymer. best, Kathryn Kathryn R. Elmer Ph.D. Candidate Department of Biology, Queen's University ------ We have a 3130 (1 year old) and probably run 10 samples a week. I had the same question you did, so I just tried it. I get great sequence up to 2.5 weeks out. I have not tried it beyond that but that cuts your polymer cost in half right there. I also aliquot polymer - 7mls into two or three bottles. Put one on for two weeks and keep the others in the 4oC. AB says leaving polymer on for more than a week will decrease the life of the capillary array. But since we run a 4 cap array which only costs ~$500 and a bottle of polymer is $360 I figured it was worth it. We are one year in and so far have seen no loss of quality due to array degradation. Hope that helps. Jeremy Jeremy Ward Assistant Professor of Biology Department of Biology 353 MBH Middlebury College ------ We have a new 3130 (4 cap array instead of the 16 cap array) that is virtually identical to your instrument. We face a similar issue here at ISU. What I have been doing is scheduling a run once a week. I still average around 30 samples a week, so I use a plate every other week. In between runs, I have the POP7 in the fridge, the water bottle on the instrument, and the array still mounted. I defrost enough HiDi for my runs before loading the plate. (I have aliquoted and frozen enough HiDi in PCR tubes for individual array runs, so I use 4 x 10 ul + 10 ul extra per tube. But you should probably have frozen 170 or 175 ul tubes for your array size.) Then I make the buffer, load the plate and refill the polymer before running. Hopefully you won’t have bubble problems, which will suck through a lot of polymer in a hurry! At the end of the runs, I store the polymer in the fridge and turn off the instrument. I have not had to toss a 7 ml bottle of POP7 yet due to expiration. We go through it fast enough. You are probably going to be in the same boat. Each time you replenish the polymer, you eat some up, so try to regulate this if you can. Plus if you have one day of running the instrument, followed by another, you won’t have to change buffer or replenish the polymer at all. Back to back days of operation has served us well. Good luck. Let me know if there is anything that I can help with. Thanks. Andy Andrew M. King BMB Instrument Technician Illinois State University ------ Hi Dan, I can't comment on the regents issue, but I can comment on idle capillary machines. Capillary sequences like to be run. Run all the time. As long as they are running they are happy and when they are running well, they will keep running well. In my experience, they do not like to sit idle. This may have changed with the new generation of capillary sequencers, but the early 310 and 3100 definitely did not perform well after being idle. Cheers, Paul I run a 3100 for a single lab here at UCB - i have little experience with a 3130 but i suspect everything's the same; Don't believe a word abi tells you. The best advice to give is to keep the machine running as consistently as possible - everything (even the computer) seems to run much more smoothly this way. But if you encounter long periods of downtime, it still isn't necessary to follow ABI's recommended schedules. Polymer will last much longer than they advise (i've gone more than a month without changing polymer, without dirty data). I've been able to use capillaries for over one thousand runs, and they can be stored off of the machine long term (months) with little affect, provided you keep the ends stored in buffer. Many companies now sell equivalent run buffers for a much lower cost - i buy from Amresco (www.amresco-inc.com, cat# K249-1L). Finally, unless you can visably see charring (from arcing events) in the polymer channels of blocks, you should never replace them. I frequently find charring in the lower block, and have been able to perform runs without replacing the block, and the data did not seem at all affected. I haven't even cleaned the blocks or syringes in over a year... Amy Smith Environmental Science, Policy and Management UCBerkeley ------ Dan, I am in a similar situation, so please post what you find out. I know that Amersham sells ABI compatible reagents that are supposedly less expensive. I still haven't tried them out and have been trying to get some free samples out of them to make sure that it works. Best wishes, Miriam Barlow ------ We do not use our ABI3730 sequencer to the extent that many larger departments do; we only have a few labs that utilize the sequencer - all on an irregular basis. The AB polymer we use (POP7 - Cat# 4363929) typically has an expiration date of about 3 months after the arrival date (I don't know which polymer your 3130 uses) . Our department typically does not come close to going through an entire bottle within the time frame of 3 mo. Thus, at $990.00/bottle, this is quite a waste of money. In the past, to extend our polymer life, we have kept only a portion of the total volume of the polymer on the sequencer, and the rest at 4C and just refill the bottle on the machine before it gets too low; this seems to extend its shelf life. We had one bottle last about 8-9 months with no sequencing problems. We found that when a bottle finally "goes bad" the sequencer will stop sequencing runs before getting to the "data collection" due to an "unstable current" (due to too much urea precipitating out); at this point, it's time to buy a new polymer bottle. The bottle we have on our 3730 right now is already 2-4 weeks "past due" and is not giving us any problems...yet. -dave hollis ------ What we did to solve this HUGE cost is to replace the "new" POP by an "old" POP when the machine is off. The polymer has a much longer life in the fridge! Another possibility, that we also use, is to aliquot the polymer in smaller volumes, but you have to be careful not to allow the polymer reach the end while runnign or you will spend a lot of polymer to eliminate bubles. Good Luck Walter Walter A. Boeger, PhD Laboratory of Molecular Ecology and Evolutionary Parasitology Grupo Integrado de Aquicultura e Estudos Ambientais (GIA) Universidade Federal do Paraná ------ for fragment analysis, you can use polymer that has been on the instrument for more than 2 weeks without too much adverse effects (your peaks won't be as sharp, but if you're not multiplexing too much, it shouldn' t be a problem). However you'd better change the buffer more often, like every 5 days. You can also save polymer by manually pushing the reserve syringe to the end (we are working with a 3100, it stops when there is still 0.5 mL of polymer). You can also save on size standard - it is recommended to use 0.5 uL per well ; we use 10 times less (even though we have a strong signal, a lot of PCR products being loaded, it works fine). -- Antoine Barrière Laboratoire Évolution du développement des nématodes Tour 43, 5° étage Institut Jacques Monod dbs4a@cms.mail.virginia.edu