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Registered: 09/12/18 Posts: 77 |
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Hello all,
Not sure if this fits in 'advanced', but I have taken interest in DNA barcoding and have some questions regarding interpreting my gel electrophoresis results. I have the minipcr blue light led transducer which boasts "observe band separation within <5 minutes" but some protocols using more 'professional' lab equipment say to run the gel for 20 minutes. The bars seem to start off clear but kind of fade out over the period of 20-30 minutes. I should mention I am not a classically trained biologist but am interested in learning. Here is my first run with photos take over time, samples are in lane 1-8 and a DNA ladder in the 9th lane: Here are my second results, I recorded exact times. samples are in lanes 1-8 and DNA ladder in lane 9: 3 minutes I guess my question is which (if any) of these samples are good enough to send off for sequencing? Im not super impressed with the DNA ladders. Any feedback, troubleshooting advice, or other info about these results you can share is appreciated. Thanks all.
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Pumperfunkfunkerpump Registered: 06/21/20 Posts: 42 Last seen: 3 years, 25 days |
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Not half bad! Are you doing all of this with a home DNA-extraction & purification setup? If so, frickin dynamite, that's awesome.
What you're looking for in a good electrophoresis result candidate to further purify and sequence is a radiant band with clear separation from its preceding streak. I wouldn't spend my time or resources trying to cut out any of these lanes to send out for analysis. Some questions to consider for your next go-around: 1. Are you using traditional PCR to amplify your DNA? If so, what restriction enzymes are being used to cut that sequence? 2. Are all of your reagents stored properly? You're never going to get good results if your materials are subjected to weak storage conditions or are too old. 3. Did you use enough dye? IIRC it should be a 1:10 ratio of visualization dye to DNA sample. -------------------- “No one is born hating another person because of the color of his skin, or his background, or his religion. People must learn to hate, and if they can learn to hate, they can be taught to love, for love comes more naturally to the human heart than its opposite.” - Nelson Mandela
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Registered: 09/12/18 Posts: 77 |
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Thanks for taking a look and taking the time to respond! And yes this is all a DIY setup in my home. I can link the protocol im using if that will help things along. I understand that I am seeking a clear radiant band but I guess I am asking at what time after running the gel is the band supposed to be clear? As you can see, some of them start off pretty crisp but fade out over time (at different rates). If they are clear and crisp at 5 minutes are they good? or should they be crisp at the 20 minute mark?
Also, why would I cut anything out of the gel? I end up with 28ul of PCR product and use 7ul to run the gel. Isn't the gel process just to check if the amplification worked so I can send of some of the remaining PCR product for sequencing? And are you saying every sample in every lane on both gels are no good? And do you think the my issues are from extraction, amplification, running the gel or a combination? To answer your questions, 1. Yes, as far as I understand the PCR process I use is a conventional PCR process. I use my NaOH extracted template DNA, ITS forward and reverse primers, Taq polymerase master mix, and distilled water to run the PCR. Then after PCR, mix with loading dye and run the gel. I don't think I am using restriction enzymes. What role do they play in the PCR process or sanger sequencing? Is it required? Im not sure why I would need to cut the DNA at any point. Don't the forward and reverse primers make it so the ITS region is the section that is amplified? Aren't restriction enzymes mostly used for synthetic biology? 2. My reagents are stored in my freezer. They are less than 3 weeks old right now. The package they came in said to store at -20C but like I said, I am doing this in my home with my own funds. At this point, I do not have access to a freezer that can go to -20C so they are stored in my kitchen freezer (around -11 to -13C) which is the best I can do right now. Do you think they're toast? 3. I used 2ul of dye to 7ul of PCR mix. Thank you again for your input. it is greatly appreciated!!
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Pumperfunkfunkerpump Registered: 06/21/20 Posts: 42 Last seen: 3 years, 25 days |
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Definitely share the protocol - it's been about 4 years since the last time I ran samples on Agarose, so forgive me if my memory's a little foggy with exact timings/concentrations
Quote: Doesn't mean they're good, most samples running on a 1.5-2% agarose gel take around 30-40 minutes to complete (depending on voltage). The fluorescent mixture hasn't had time to dissipate and separate in 5 mins, which is why it appears so radiant. Quote: 1. You cut the samples out to get sequenced if you're running multiple samples in the same gel. I've never run 8 samples of the same sequence in a gel, idk if that's what you're asking. 2. Yes. 3. Yes, don't send these out and waste your money. Go for another run and try getting better results. 4. Not sure, idk what reagents/protocol you're using. Quote: Yup that's traditional PCR. I would use RNAse-free water instead of distilled though. You'll get consistently muddy results if you're using non-lab-grade ddh20. Quote: Prime example of my memory hobbling things together - used to cleave DNA sequences from 1 organism and transform -> another org and do PCR on the recipient org. Your taq pol and primers are what ya need ![]() Quote: Nah they're good. -------------------- “No one is born hating another person because of the color of his skin, or his background, or his religion. People must learn to hate, and if they can learn to hate, they can be taught to love, for love comes more naturally to the human heart than its opposite.” - Nelson Mandela
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Registered: 09/12/18 Posts: 77 |
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Thanks for the info!
Quote: No worries, last time I did this was in high school biology 10 years ago. Unfortunately back then I was a bit of a rascal and didn't give a shit about school so I am starting fresh. I am mostly looking at protocols that are geared more toward citizen science/DIY bio because I feel like the available resources are more realistic for my situation. Here is the protocol I followed: https://docs.google.com/documen Quote: I am running 1% gel if that makes a difference. I believe this is dictated by the size of the ITS region. And so do you think the bluegel product is just marketing out their ass when they make claims like "Observe DNA separation in as little as five minutes"? Will the blue light LED at 48V going to run with any significant difference from a more traditional higher voltage usually (100v) UV gel electrophoresis set up? The Counter Culture Labs protocol mentions in the electrophoresis that " If you leave it for 30 minutes or longer, you will lose your results" which might explain why my bands fade out over time, but your point that it appears so radiant in the beginning because it hasn't had time to separate makes a lot of sense to me. The fact that anything is running and radiating in the gel means that some DNA has been extracted and amplified? Are the inconsistencies in my results from the different samples due to inconsistencies in my extraction process leading to varying levels of success and/or varying levels of contamination? Quote: Each lane is from a different sample. After PCR, I have 28ul of PCR product. I put 7ul of PCR product and 2ul of dye from each sample into each well, leaving 21ul of PCR product left behind in the PCR tube. I am under the impression that I would just send off some of the remaining 21ul of PCR product left in the tube for sequencing instead of trying to cut out what I ran through the gel. Is it better to cut it out and purify it? Quote: The protocol I use and the Counter Culture Labs protocol both used bottled distilled water from the grocery store. William Padilla-Brown uses aquafina. They get successful results but I'll look into buying lab grade RNAse-free water. Quote: excellent. Quote: I am absolutely going to try again but I'm not really sure what to do differently. I think overall my results were better the second time through but that could have just been because the samples used were different. It feels wasteful to do the process over the exact same way if you say its not working. Any suggestions for troubleshooting or moving forward on the next round? Thanks again for taking your time to answer my questions. I taught myself PCR through reading over the past month or so and don't have anyone in my direct personal life who has experience with this process for instruction or to ask questions. It is truly appreciated.
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Registered: 04/22/18 Posts: 353 Last seen: 3 months, 15 days |
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Cool, glad to see another person getting into this. I didn't see you mention what voltage you are using. Just a reminder, running too high a voltage will result in more U-shaped bands while too low a voltage will tend to make blurry bands.
If I were you, I'd try and fine tune your process and voltage by getting the dna ladder dialed-in. If you can get nice clear bands with just the ladder, then you can rule out a few big variables from what might be affecting your other samples. Most ladders should also have some protocol parameters suggested. AddGene also has a really good overview with videos for running gels that you might also find helpful: https://www.addgene.org/protoco What gelbox you using btw? I'm looking for one myself.
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Registered: 09/12/18 Posts: 77 |
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Thanks for contributing!
Quote: I am using the bluegel integrated electrophoresis and visualization system from minipcr. It runs on just 48V. It is not adjustable and the person who made the protocol I use has the same one so I figure this eliminates some variables. Quote: This is a great suggestion, thank you! The DNA ladder was in the far right lane on both samples and I wasn't impressed with it in either run. I am using the ladder from the odin ( https://www.the-odin.com/dna-la Quote: excellent link, thanks. They mention "A typical run time is about 1-1.5 hours, depending on the gel concentration and voltage." !! That is significantly longer than other sources I have read. The Counter Culture Labs protocol says "If you leave it for 30 minutes or longer, you will lose your results". These statements seem contradictory... not really sure what to do with this. To be honest, I've just been letting it run and looking at it periodically because with the bluegel box, the transilluminator is built in, allowing you to view it at any point while it runs. At the 1-1.5 hour mark, I would have not results left to look at. I am wondering how different the bluegel system is from a more typical electrophoresis system. Quote: I am using the minipcr bluegel Integrated electrophoresis and visualization system. I didn't buy the whole minipcr kit because some of the other equipment like the thermalcycler can be found much cheaper on ebay. At the time the bluegel system was on sale for $250. I am not sure what it usually costs but I added up the cost of the trays, a UV transilluminator, electrophoresis power supply, etc plus the shipping for all the individuals parts and it was more expensive at the time, so I went with Bluegel. But shop around, there may be better deals now, ebay fluctuates. I do like how the system is compact and integrated. the whole system fits in an 6"x10" box with room to spare. Comes with a neat little prop up box to take nice photos and document the run. Just curious, when you look at these results what are your impressions? Total trash, good enough to sequence, or somewhere in between? contaminated? I know they are not perfect but I am considering spending the 7 bucks or whatever to try and have one of the better results sequenced just to see what happens. I honestly feel like I am over complicating this process but I am invested to the point where I am determined to make it work. Thank you for taking your time to respond, I appreciate it
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Registered: 04/22/18 Posts: 353 Last seen: 3 months, 15 days |
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Yea, 48V seems low from what I've read. The CC labs protocol recommends 100V but really it depends on the distance of the gel the bands will be traveling. AddGene also recommends 80-150V. You're using about half of at least 2 other published protocols - so something to consider in terms of variables.
Quote: If you keep going with the 48V, I would just greatly expand the length of time you run it, observing closely for when it begins to degrade in quality. Lower voltages take longer but should give you better clarity. Plan to waste a few trials so that you can intentionally go over the allotted time window in order to find the optimum. Quote: The Odin's ladder also references using 2% gel which isn't to say your 1% won't work, but I'd try and imitate a known working protocol before deviating myself. Do you have a camera? You could try to film a timelapse over 2 hours to help you analyze the bands. I believe an iPhone and an app could do this - just need to work out a good angle and lighting. Also, AddGene suggests using the 10% rule: Quote: Addgene also says "Note: Make sure to use the same buffer as the one in the gel box (do not mix different buffers and do not use water)." TheOdin ladder says "This ready-to-use product is premixed with 1×DNA loading buffer." Are you using different loading buffers? That could be another variable. And finally, you'll want to rule out any issues with how you're loading the ladder: Quote: Quote: I haven't run one myself so I'm not one to ask, BUT I've been reading and watching tutorials like crazy and I can definitely say that you aren't getting the kinds of clear bands that one should expect. If you were to send off for sequencing, you'd be gambling imo. If it were me, I'd try and get this part working before sending off for sequencing because it's such an important skill to learn and it is actually designed to save you time/money anyway. Keep plugging away at this until you get a more confident result and then you can send off your sample and divert your energy to the sequencing knowing that everything else is good up to that point. In my mind, the variables you can play with are: * Voltage * Runtime * Loading volume * Loading technique * Buffer compatibility Thanks for the info on your rig. I'm going to look into it and get one going soon. PM me if you want to stay connected and support one another. Keep us updated!
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Registered: 09/12/18 Posts: 77 |
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Thanks for the feedback! This is great.
Quote: As mentioned, the 48V is not adjustable. The protocol I used ( https://docs.google.com/documen Quote: So I just measured the gel tray and it is 5.75cm. With 5-8V/cm, 5.75x5=28.75 and 5.75x8=46, looks like it should be operating at 28.75-46V. I believe the ITS length varies by species but I don't think it will be any more than 2kb. Quote: I love the idea of not deviating but the protocol I have been following does not use a ladder. The CC labs protocol runs 1% gel with this ladder. But your point about mixing buffers is an excellent one that has me thinking. So, the CC labs protocol and the one I follow calls for the same loading dye, but the CC labs protocol uses TAE in the agarose mixture as well as to submerge the gel, while the protocol I followed uses TBE for both the agarose mixture as well as to submerge the gel. TBE is recommended by bluegel over TAE and even the CC labs protocol mentions that TAE might not be the best and that borate might be better (TBE is Tris, Borate, and EDTA). Do you think addgene might just be talking about not using a different buffer in the agarose mixture than the one that gets poured over the gel? From the product description of the loading dye that both protocols use (CC labs and the one I follow) have no mention of TAE or TBE. It says it is a mixture of two dyes, SDS, EDTA, and Ficoll. I wonder what the buffer in the ladder is, I would like to believe that they would mention that in the product details if it was important but maybe that is wishful thinking. Quote: This was the train of thought I was using on the second run by taking photos at 3 minutes, 5 minutes, 13 minutes, and 20 minutes. They absolutely change over time which is what really made me start this thread. If CC labs protocol says "If you leave it for 30 minutes or longer, you will lose your results." but Dr. Whitethumb above made the point that "The fluorescent mixture hasn't had time to dissipate and separate in 5 mins, which is why it appears so radiant.", when are you supposed to call it? 5 minutes being too soon and 30 minutes being too late, am I shooting for anywhere in between? 10 minutes looks very different than 20 minutes. Bluegel advertises "Observe DNA separation in as little as five minutes" and the protocol I follow says "You should see a band on each lane after about a minute or so. If you don’t see a band the PCR didn’t work for that sample. Let it run for a while." This kind of suggests that it might be fine but doesn't explain why my ladder didn't come out clean. I think at 2 hours my results would be gone altogether. I'll take more photos with less time in between and/or make a time lapse next time around for more specific documentation. Quote: Oh I totally agree with you there. I was kind of thinking it might be a way to troubleshoot the process as a whole. If it sequenced fine then that would mean that my extraction and amplification were successful but my gel electrophoresis needs work. But alas, I believe you are correct about the getting the ladder to come out properly as it is known DNA. If that doesn't work than my samples won't either. Quote: Good idea to make a list of variables. I think I'll play around with loading volume and loading technique next time around and see if anything works. I can't change voltage and I have just been running it until it disappears. I can view it while it runs so I haven't really seen a need to stop the run at any point if I have been documenting it as it goes. Run time is kind of the variable I'm trying to pin down. I'll do more reading on buffer compatibility in the meantime but I'm going to see if I can get it to work with what I have before I buy anything else. I'm doing this with my own funds so I try not to buy anything unless it is necessary. I'll think about other possible variables as well. Quote: I would love to stay connected and support one another. I really appreciate you taking the time to try and hash this out with me. I am happy to help you out in any way I can when you get your system going. We can move this discussion to PM if you want, I am down to keep it public as well in case anyone else down the road is working on this with similar questions. I believe citizen science is a great way to contribute to the world, especially in such an under-researched field as mycology, and I think supporting one another is a critical part of this movement. I plan to keep this thread updated for sure. I am going to try another run soon, I don't have time tonight but maybe tomorrow. The whole extraction/amplification process takes a long time (the PCR part alone runs for 2-3 hrs) but I might just tinker with the gel electrophoresis part using the samples from last run or just the ladder to try and see how results can vary. Thanks again!
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Registered: 04/22/18 Posts: 353 Last seen: 3 months, 15 days |
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Quote: Makes sense. I was suggesting that if you had another power supply laying around, you could try and hook into the electrodes, but it doesn't sound like that's the case. I tend to agree with your other points about it being designed to work at the supplied voltage, all else being equal. Quote: Digging into their FAQ about this statement, it says: Quote: I think they're just saying that results will begin to develop in 5 minutes but it will take 15-20 mins to complete. Quote: Exactly - just need to ensure they are the same. It also says that you can use TBE instead of TAE so that's also good to know. Might shoot Odin an email to ask about the buffer used in the ladder. Sounds you have a good handle on next steps. It'll be nice if you can get it working since I definitely plan to come pick your brain once I go hands-on in the coming weeks. ![]() Quote: Absolutely brother. Right there with ya. Reach out anytime. You can also follow me on the gram @everymanbio or my site. I'm networked with a few different like-minded folks here and I'm actively working to put together a fungi genetics project we can all collaborate on together.
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Pumperfunkfunkerpump Registered: 06/21/20 Posts: 42 Last seen: 3 years, 25 days |
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I'm glad someone else jumped in and was able to shed some insight! Sorry I've been away for a few days, life got kinda busy
![]() The protocol you're using looks sound. I agree with all of the statements outlined by PTree Quote: Keep on tinkering with those variables and you're bound to get better results. Quote: When I was apprenticing in a lab, I was instructed to always send out agarose samples for sequencing. The reasoning that was given to me was that gel is a better protectant than free-flowing liquid. Makes sense to me Quote: Yeah, more of a sales pitch than anything. You can OBSERVE separation in five minutes, not get RESULTS in five minutes ![]() Really dope you guys are interested in this side of the science behind mycology. PTree - checked out your site and would love to contribute in some way that's helpful. Let me know when you've got parts of your culture library for sale, would be happy to support the cause Please keep the thread updated with pics of the new run whenever you get around to giving it another shot Jawns! -------------------- “No one is born hating another person because of the color of his skin, or his background, or his religion. People must learn to hate, and if they can learn to hate, they can be taught to love, for love comes more naturally to the human heart than its opposite.” - Nelson Mandela
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Registered: 04/22/18 Posts: 353 Last seen: 3 months, 15 days |
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Quote: Right on. I'm working on getting some of those up for sale actually. If you shoot me a PM with a reminder, I'll create a special coupon for you to get some freebies, so long as you don't mind sharing feedback on the order process when it's up.
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Registered: 09/12/18 Posts: 77 |
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I took another run through the gel electrophoresis process and I'll share my results to keep the thread updated in case it helps anyone.
Quote: Interesting! Makes sense to me too. The places I'm looking at for sequencing (Genewiz, Mclab, etc) have pretty specific requirements on how they want their samples and they just want the PCR product from what I have read. But your point makes me wonder how to store the PCR product between the PCR process and sending it off for sampling. Is it shelf stable? Is one supposed to put it in the fridge? the freezer? I've read you can autoclave DNA extractions but the raw extraction might have different properties than the PCR product. Either way, I didn't have time to do a whole extraction and amplification process so I just used remaining PCR product from a week ago or so that sat out on my desk (in no AC, 78-85F) and it seemed to work out ok. So this time I played around a lot with loading technique. The gel casting tray has two spots for combs so I decided to try one with two combs. This helped narrow down the time question because I imagine it is designed to be read before it runs into the next set of wells or off the bottom of the gel. I think I need a more accurate scale for weighing out agarose too, which is another variable. I played around with loading technique a lot here, not being afraid to waste a run to see how things work. Some of them I kinda stabbed it into the gel, some of them the pipette tip was barely in the buffer. Stabbing it in the gel makes for very poor results. I think the best results came from keeping the pipette perfectly upright and not going down to the second stop on the pipette to keep from producing bubbles. Also having the pipette tip in the buffer just above the well and letting the PCR product/loading mix sink into the well seemed to produce the best results. 5 mins As you can see a lot of them are jacked up but thats because I was really messing around with loading technique. Ladders are in wells 5,6,and 7 in the top row and 1 and 7 in the lower row. by the end of the run the ladder had separated pretty clearly in the more successful lanes to the point where I could actually measure the size of the bands of PCR product. excuse how blurry some of the photos are, there was some condensation on the top of the box. Im going to try the full process over again sometime this week. hopefully I'll get something I can send of for sequencing because I want to learn how to use that data. Feedback and ideas are always welcome and appreciated. Thanks again Ptree and Dr. Whitethumb!
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Registered: 04/22/18 Posts: 353 Last seen: 3 months, 15 days |
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Wow, that looks much better!
Quote: That makes a lot of sense. You want the dna to be suspended in the buffer so that the charge can be more easily conducted through the negatively charged molecules. Jabbing it in the gel would certainly impede the ability for the strands to move freely through the solution. Top row lane #7 looks great. Looks like optimal time is around the 35 min marker. Maybe a little longer if you want to get more separation on the shorter bands. I can help you generate phylogenic trees and publish your sequence to GenBank when you get to that point. So happy for you brother. Keep it up! Can't wait to hear how the sequencing goes.
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Pumperfunkfunkerpump Registered: 06/21/20 Posts: 42 Last seen: 3 years, 25 days |
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Quote: Not exactly sure man, it was a fairly quick turn around time whenever I sent samples off for sequencing, like same-day or next-day shipment. The joys of a professional laboratory. I'd err on the side of caution and keep your product in a fridge. At the very least it will slow degradation. Not sure if the DNA fragments would be freezer-stable if not snap-frozen with liquid nitrogen first. Quote: Brilliant! Much better job this time around. Keep up the great work dude!
-------------------- “No one is born hating another person because of the color of his skin, or his background, or his religion. People must learn to hate, and if they can learn to hate, they can be taught to love, for love comes more naturally to the human heart than its opposite.” - Nelson Mandela
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Registered: 04/22/18 Posts: 353 Last seen: 3 months, 15 days |
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Hey Jawn876, any updates? Were you able to successfully run PCR on your samples and send them out for sequencing? I've got my barcoding lab setup and reagents in-hand. I'll probably attempt my first few samples later this week. Curious to know which lab you used for sequencing and what the experience was like order, sending them in and getting back the data. Hope all is well!
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Elephant in the Woods Registered: 08/18/19 Posts: 212 Loc: N 42nd Parallel Last seen: 2 months, 11 days |
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Registered: 04/22/18 Posts: 353 Last seen: 3 months, 15 days |
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I recently posted about my first PCR & DNA barcoding experience.
Here is photo of one of my first gel results. There's a timelapse video in aforementioned link.
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Registered: 09/12/18 Posts: 77 |
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Thats awesome man!! Sorry I missed your last post, I was out of town for about 6 weeks and everything got put on hold so I unfortunately haven't sent anything out for sequencing. I have done a few successful PCR runs though and have some samples ready to send out. I got a little lost in the sequencing order forms. Nice work though! If you dont mind sharing, did you send out your samples for sequencing? What lab did you use? Did you get results back for all your samples in that gel? Is that a ladder in the far left lane? Really cool to see you having success with this! I would love to hear all about it. Cheers
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Registered: 04/22/18 Posts: 353 Last seen: 3 months, 15 days |
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My samples are en route to MCLAB (https://www.mclab.com/) for sequencing. Should get the results back via email mid next week. The form was a mess so if you need help, give me a shout.
Yea, the first lane is the ladder and I've been having some issues with it. The first time, I think I just loaded it incorrectly. The 2nd time, I loaded it first while I loaded all of the other wells and by the time I was ready to flip the switch, it looked like it was starting to bleed out of the well. So I think both times, my loading technique probably messed it up. I'm not even sure a ladder is necessary for single gene extraction though. As long as there is one solid band and all the samples show the band in the same position, that's pretty good indication of a positive reaction. BTW - I used Sigrid's protocol that you linked above and ended up interviewing her for my podcast on YouTube. She's great and she also was the one who helped me figure out the order form.
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