Alternative Sources of BigDye XTerminator

Dynamac · 05-07-2012, 06:32 AM

Does anyone know of alternatives to Life Technologies for this reagent? Since their acquisition of ABI, our cost has nearly doubled, and the costs for other reagents have increased as well.

Thanks!

Barry

Elcannibal · 05-08-2012, 01:10 PM

Quote:

Originally Posted by Dynamac

Does anyone know of alternatives to Life Technologies for this reagent? Since their acquisition of ABI, our cost has nearly doubled, and the costs for other reagents have increased as well.

Thanks!

Barry

I don't think you will get an answer for this because:
1- BDT is the biggest cash cow for Life Tech and always was (brings back memories of the acronym ABI, good ld days I must say).
2- It will keep going up ie stock market, gold watches private jet.
3- IP, is what drives innovation, without it, no funding, unless of course it is for a cure, then it would likely be about 100x the price.

You should do the following, order bulk, order within 3 weeks of a final quarter (end of March, June, Sept, Dec), tell your rep you will not order before the end of the month unless there is significant discount. Final solution is to move over to ET Terminator chemistry from GE. Pretty similar and much lower in cost but expect a support embargo from Life.

Don't expect more than 15%, BDT is the most controlled ingredient at Life.

han526 · 05-26-2012, 12:39 PM

What I found is that once you optimize the conditions (mostly robotic handling), the magnetic beads purification works very well. Use less Dye terminator, clean and long reads. You may try PureSEq or CleanSEQ. Xteminator is propriatory. Life tech has a monopoly there.

Dynamac · 05-29-2012, 02:27 AM

Thanks all, for the input. Life offered me 12%. I'll have to have discussions with our client about the alternatives.

athensugadawg · 09-15-2012, 03:55 PM

when optimized on a robotic platform, e.g Beckman, Hamilton, Tecan (can use manually as well), will allow you to effectively take your BigDye dilution down to 1:16 for shotgun and 1:32 for PCR product. Cost per prep is quite favorable as well....Omega Bio-tek has a product that could possibly fit your needs.

MrGuy · 09-16-2012, 10:14 PM

I thought the guy was talking about xterminator and not BigDye? Agencourt makes a competitive product.

The best way to decrease the cost of xterminator is to run a 5-10 ul BigDye reaction vs the 20uL you are likely running now.

Dynamac · 09-17-2012, 07:20 AM

I was talking about XTerminator. We doing a 1/8 reaction at 10 µL volume now.

MrGuy · 09-18-2012, 06:18 AM

Same here, but we run 1/4 reaction with a 25 minute sanger cycling.

Dynamac · 09-18-2012, 07:00 AM

While we're on the subject, another question about the 3730xl DNA Analyzer. What effect does oven temperature have on separation? If I lower the temp, will the separation be better?

MrGuy · 09-18-2012, 11:29 PM

Consider it this way...
You have a signal moving past a camera with a finite number of pictures it can take over certain time. To increase resolution, you would want more pictures per signal, right?

Thus, you can increase resolution by slowing the flow with:
1) longer path (capillary)
2) lower voltage (slower migration)
3) thicker polymer (I just stick with pop7 so I don't have to change anything)
4) colder oven (thicker polymer)
5) cold window (3130 has a heated window, can't remember if the 3730 has it).

So, basically just find a slow default run module, shorten or lengthen the time as it fits your product, then make some fine adjustments with your oven temp if you prefer.

Dynamac · 09-19-2012, 02:27 AM

Thanks, MrGuy.

I don't think the 3730 supports anything other than Pop7, we're stuck with a 50-cm array, so we'll have to try to vary the voltage and oven temperature. We have short products, so we may never resolve them.

Barry

MrGuy · 09-19-2012, 06:08 AM

How short is short? I used to sequence realtime products every now and then on a 36cm with the UltraSeq module/pop7

Dynamac · 09-19-2012, 06:58 AM

100 bp! These are for a client. They may be qPCR products, I'm not sure.

MrGuy · 09-20-2012, 03:39 AM

Throw on M13 tails and sequence through the primer. You'll gain a few bases that you need.

pmiguel · 09-20-2012, 04:22 AM

The oven temp is designed to keep the product strand denatured so you end up with less product strand compression events.

For your application you might want to abandon big dye for the ancient "dRhodamine" chemistry. Big Dye's bulky fluors contribute to the lower quality of sequence very close to the primer. (You probably need to run a spectral for it, but I am not sure.)

Keep in mind that the low quality of the sequence may be a result of those PCR products being contaminated with primer dimers, not some issue with migration rates.

I would think that the 3730XL could handle other polymers. Might want to post on the ABRF forum. Higher density of first-gen experts there. Also, check with ABI tech support. Some times they come through.

--
Phillip

pmiguel · 09-20-2012, 04:30 AM

Quote:

Originally Posted by Dynamac

Does anyone know of alternatives to Life Technologies for this reagent? Since their acquisition of ABI, our cost has nearly doubled, and the costs for other reagents have increased as well.

Thanks!

Barry

Ethanol precipitation. But you need to seriously cut back on your Big Dye because EtOH isn't as effective at removing persisting reactants (dye blobs). Also, we generally use less than 2 volumes of ethanol to reduce the final salt concentration and reduce the predominance of short products. May not be as critical if you load in formamide -- we always do water loads for the higher signal strength. Oh, you also need a centrifuge, so it may not be ideal for your circumstances.

--
Phillip

Dynamac · 09-20-2012, 05:05 AM

Thanks, all.

Vinz · 09-24-2012, 01:30 AM

BD V1.1 is supposed to be better for the short products than BD V3.1
We use POP6 on one of our 3730XL for reading early. Using BD V1.1 and Pop6 reading from base 20 onwards is no problem. If you correct it manually, you could read from base 1 often. Caveats: Pop6 is only sold in very small aliquots and with a huge price tag.
If you are interested I could share the settings.

Dynamac · 09-24-2012, 02:59 AM

I was under the impression that Pop 6 would not work on the 3730, but I'm glad to hear otherwise. If you could share the settings with me that would be great!

You can send a private message if you'd like, or just post them here.

Thanks, Vinz.

Vinz · 09-24-2012, 04:51 AM

We got a patch from ABI to allow for POP6. If I recall correctly the main thing is a new POP6 mobility file to be selected in the "analysis protocol". But I think this is not required.
Oven temperature is set to 55°C
PreRun and Run Voltage at 15kV (13kV gives a little better resolution, but takes longer)
160ms readoutime (first and second)

I would recommend a new spectral calibration after changing the POP.

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05-07-2012, 06:32 AM	#1
Dynamac Member Location: Cincinnati, OH Join Date: Aug 2011 Posts: 37	Alternative Sources of BigDye XTerminator Does anyone know of alternatives to Life Technologies for this reagent? Since their acquisition of ABI, our cost has nearly doubled, and the costs for other reagents have increased as well. Thanks! Barry

05-26-2012, 12:39 PM	#3
han526 Junior Member Location: Boston Join Date: Feb 2010 Posts: 4	Have you tried magnetic beads based kits? What I found is that once you optimize the conditions (mostly robotic handling), the magnetic beads purification works very well. Use less Dye terminator, clean and long reads. You may try PureSEq or CleanSEQ. Xteminator is propriatory. Life tech has a monopoly there.

09-15-2012, 03:55 PM	#5
athensugadawg Junior Member Location: ga Join Date: Feb 2011 Posts: 1	MagBead-based DTR when optimized on a robotic platform, e.g Beckman, Hamilton, Tecan (can use manually as well), will allow you to effectively take your BigDye dilution down to 1:16 for shotgun and 1:32 for PCR product. Cost per prep is quite favorable as well....Omega Bio-tek has a product that could possibly fit your needs.

05-29-2012, 02:27 AM	#4
Dynamac Member Location: Cincinnati, OH Join Date: Aug 2011 Posts: 37	Thanks all, for the input. Life offered me 12%. I'll have to have discussions with our client about the alternatives.

09-16-2012, 10:14 PM	#6
MrGuy Member Location: earth Join Date: Mar 2009 Posts: 58	I thought the guy was talking about xterminator and not BigDye? Agencourt makes a competitive product. The best way to decrease the cost of xterminator is to run a 5-10 ul BigDye reaction vs the 20uL you are likely running now.

09-17-2012, 07:20 AM	#7
Dynamac Member Location: Cincinnati, OH Join Date: Aug 2011 Posts: 37	I was talking about XTerminator. We doing a 1/8 reaction at 10 µL volume now.

09-18-2012, 06:18 AM	#8
MrGuy Member Location: earth Join Date: Mar 2009 Posts: 58	Same here, but we run 1/4 reaction with a 25 minute sanger cycling.

09-18-2012, 07:00 AM	#9
Dynamac Member Location: Cincinnati, OH Join Date: Aug 2011 Posts: 37	While we're on the subject, another question about the 3730xl DNA Analyzer. What effect does oven temperature have on separation? If I lower the temp, will the separation be better?

09-18-2012, 11:29 PM	#10
MrGuy Member Location: earth Join Date: Mar 2009 Posts: 58	Consider it this way... You have a signal moving past a camera with a finite number of pictures it can take over certain time. To increase resolution, you would want more pictures per signal, right? Thus, you can increase resolution by slowing the flow with: 1) longer path (capillary) 2) lower voltage (slower migration) 3) thicker polymer (I just stick with pop7 so I don't have to change anything) 4) colder oven (thicker polymer) 5) cold window (3130 has a heated window, can't remember if the 3730 has it). So, basically just find a slow default run module, shorten or lengthen the time as it fits your product, then make some fine adjustments with your oven temp if you prefer.

09-19-2012, 02:27 AM	#11
Dynamac Member Location: Cincinnati, OH Join Date: Aug 2011 Posts: 37	Thanks, MrGuy. I don't think the 3730 supports anything other than Pop7, we're stuck with a 50-cm array, so we'll have to try to vary the voltage and oven temperature. We have short products, so we may never resolve them. Barry

09-19-2012, 06:08 AM	#12
MrGuy Member Location: earth Join Date: Mar 2009 Posts: 58	How short is short? I used to sequence realtime products every now and then on a 36cm with the UltraSeq module/pop7

09-19-2012, 06:58 AM	#13
Dynamac Member Location: Cincinnati, OH Join Date: Aug 2011 Posts: 37	100 bp! These are for a client. They may be qPCR products, I'm not sure.

09-20-2012, 03:39 AM	#14
MrGuy Member Location: earth Join Date: Mar 2009 Posts: 58	Throw on M13 tails and sequence through the primer. You'll gain a few bases that you need.

09-20-2012, 04:22 AM	#15
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 1,698	The oven temp is designed to keep the product strand denatured so you end up with less product strand compression events. For your application you might want to abandon big dye for the ancient "dRhodamine" chemistry. Big Dye's bulky fluors contribute to the lower quality of sequence very close to the primer. (You probably need to run a spectral for it, but I am not sure.) Keep in mind that the low quality of the sequence may be a result of those PCR products being contaminated with primer dimers, not some issue with migration rates. I would think that the 3730XL could handle other polymers. Might want to post on the ABRF forum. Higher density of first-gen experts there. Also, check with ABI tech support. Some times they come through. -- Phillip

09-20-2012, 05:05 AM	#17
Dynamac Member Location: Cincinnati, OH Join Date: Aug 2011 Posts: 37	Thanks, all.

09-24-2012, 01:30 AM	#18
Vinz Member Location: Germany Join Date: Dec 2010 Posts: 53	BD V1.1 is supposed to be better for the short products than BD V3.1 We use POP6 on one of our 3730XL for reading early. Using BD V1.1 and Pop6 reading from base 20 onwards is no problem. If you correct it manually, you could read from base 1 often. Caveats: Pop6 is only sold in very small aliquots and with a huge price tag. If you are interested I could share the settings.

09-24-2012, 02:59 AM	#19
Dynamac Member Location: Cincinnati, OH Join Date: Aug 2011 Posts: 37	I was under the impression that Pop 6 would not work on the 3730, but I'm glad to hear otherwise. If you could share the settings with me that would be great! You can send a private message if you'd like, or just post them here. Thanks, Vinz.

09-24-2012, 04:51 AM	#20
Vinz Member Location: Germany Join Date: Dec 2010 Posts: 53	We got a patch from ABI to allow for POP6. If I recall correctly the main thing is a new POP6 mobility file to be selected in the "analysis protocol". But I think this is not required. Oven temperature is set to 55°C PreRun and Run Voltage at 15kV (13kV gives a little better resolution, but takes longer) 160ms readoutime (first and second) I would recommend a new spectral calibration after changing the POP.