Hi, here is the list of all replies I got on my question about the NED dye substitution. I am truly grateful for all the answers - they were really helpfull. It seem that it it possible to substitute NED with some other dye..I will try it out soon. best regards tomasz -- Tomasz Wilk Institute of Environmental Sciences Jagiellonian University Gronostajowa 7 30-387 Krakow, Poland *********** the question was: > Hi, >does anyone know whether it is possible to substitute NED with some other >dye when doing fragments analysis on ABI? I've read that it may work with >TAMRA. Did anyone try such substitution? >Any comments will be very helpfull... > >thank you in advance >tomasz ********** ...and the answers are: Hi Tomasz, The short answer is that the dyes you should choose depend on what sequencing machine and filter set you are using. The ABI 3730 comes with a pre-installed filter called the G5 Filter Set, which is set up for 6FAM (blue), VIC (green), NED (yellow), PET (red) and LIZ (orange, size standard). The ABI 3100 is similar, but is set up with the D Filter Set which is FAM (blue), HEX (green), NED (yellow) and ROX (red, size standard). The filter sets are pieces of software that the instrument writes during a calibration process - the emission spectra of the dyes overlap quite a lot so the 3730 tries to choose a wavelength to read each dye that minimizes the 'background' from the other dyes. For the G5 filter set VIC, NED and PET are all proprietary dyes so are only available from ABI (they are quite expensive). It is possible to substitute VIC with the much cheaper HEX but if you do this you won't be able to use NED. This is because HEX has a slightly higher max emission than VIC, which causes HEX to have spectral overlap with NED, so if you run HEX and NED together with the G5 filter you will contaminate your NED signal with signal from HEX - called "spectral bleeding" or "pull-up" (we found this out the hard way!). Of course other filter sets may exist, and it is possible to make a customized filter set, i.e. program the sequencer to read the different dyes (such as what you suggested, replacing NED with TAMRA - although we have heard that TAMRA is a particularly bad dye) at the most appropriate wavelengths for those dyes, which is something you also could try. In the end, we stuck with the dyes recommended by ABI for the G5 filter set for use on the 3730 for our own AFLP studies. We would probably recommend you do the same - the ABI dyes may be more expensive but you could potentially waste a lot of time and money trying to get the sequencer set up with different dyes (and ABI warned us that they might only be able to provide limited support if we got ourselves into that situation). Here are some links you might find useful: http://www.sigmaaldrich.com/sigma/general%20information/oligo_brochure.pdf http://www.cstl.nist.gov/div831/strbase/pub_pres/NEAFS_CEintro.pdf p. 18 http://stokes.chop.edu/action.lasso?-database=wr&-response=intranet/cores/napcore/3730FragmentAnalysis.html Heidi Meudt & Andrew Clarke ----------- Dear Tomasz, yes, you are right. It is possible to take TAMRA instead of NED. NED is something like a improved form of TAMRA. We are working on an ABI 377 and never experienced problems, when using the same matrix for both dyes. Hope this helps. If you have further questions, please do not hesitate to contact me again! Good luck and best regards, Cornelya Dipl. Biol. Cornelya Kluetsch Zoologisches Forschungsinstitut und Museum Alexander Koenig (ZFMK) Adenauerallee 160 53113 Bonn Germany Tel.: ++49-+228-9122-242 Fax: ++49-+228-9122-202 E-mail: c.kluetsch.zfmk@uni-bonn.de ----------- Hi Tomasz, we have been dealing with the same issue with a new ABI 3130xl. Several of the dyes we use on the 310 are not supported by ABI for their new machines. Their application specialist says that TAMRA labeled samples should be run separately from other dyes because of spectral overlap and possible peak "pull-up" problems. However, we have recently been running multiple dyes (HEX, 6-FAM, and Promega's CRX size standard - similar to ROX) with TAMRA and have not had any problems. You might just give it a go and see how the data look. Best regards, Rich ----------- Hi Tomasz, Various combinations of fluorescent dyes can be used with the different filter sets available on ABI sequencers. I have attached a Excel table (excerpted from ABI literature) that we use to keep track of such things - any of the dyes with a color indicated under a filter set can be used with that set. For example, filter set "C" allows use of Tamra (size standard) plus fragment sizing of products labelled with 6FAM, Hex, and Tet. Similarly, filter set "D" allows use of Rox (size standard) plus fragment sizing of products labelled with 6FAM, Hex or Vic, and Ned. Rox and Ned (using the D set) have better color separation and signal strength, so a good option if you are ordering new primers and size standard. Upgraded machines may allow use of up to 5 different dyes ("G5" and "E5"). Hope that helps! Frank Frank Cipriano ------------ Hi Tomasz, It was my understanding that the machines are standardized to specific dye sets and that NED cannot be substituted; it has to do with the emission spectra for each dye and the amount of overlap between the emissions that affect the laser's ability to detect the different dyes. Perhaps you could use it depending on the other dyes you are using. You are probably already aware that VIC can be subsituted with HEX on the 3730, if you are using that system. I would like to know what you here from other people. Amanda ------------- Dear Tomasz All of the dyes have a broad emission range and will be picked up at several waavelengths. That is why a spectral matrix is needed to compensate on ABI sequencers. HEX and VIC have similar emiisions Max, 553 and 554 so we often use HEX instead. It gives a small amount of signal in yellow as well as the green. It also results in a negative signal in red NED is 575 TAMRA is 582 PET is 595 so TAMRA would give you signal in both yellow and red on the DS33 G5 filter set. I have not tried it to see what other effects it would have but it will give you a signal Alan ----------------- thanks again for all the answers tomasz wilk@eko.uj.edu.pl