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Hi Debbie
You can sequence the PCR product directly – this is generally much simpler than cloning and sequencing. The only thing you need to do first is remove the leftover primers and dNTP. I would recommend using a PCR product purification kit (there are hundreds out there) to do this. Just make sure that you don’t use too much of the PCR product in the sequencing reaction (ie follow the bigdye kit instructions).
Cheers
Daniel Tillett
Nucleics SupportIt depends what the PCR products are stored in. If it is in water it is best to freeze the sample unless you are going to use them within a few hours.
I prefer to use 10/0.1 TE [10mM Tris, 0.1 mM EDTA, pH 8.5] to store my PCR products. The tris helps stop the solution from becoming acidic (cause by CO2 being absorbed from the air) and the small amount of EDTA doesn’t interfere with the sequencing reaction. If you use this then you can just store at 4.deg.C. I have found that stored in 10/0.1 TE the PCR products will last for years in the fridge.
Daniel Tillett
Nucleics SupportHi Larry
This is a good question. I do know that that the PE2400 had a major design flaw in regards the cooling unit. I have owned a couple in the past that have had to be fixed (I think one was fixed three time). We just sent them off to be fixed so I have no idea what the coolant used is. I guess you could try using radiator fluid – the worst that could happen is you destroy them 🙂
Daniel Tillett
Nucleics SupportIt sounds like it might be a signal spike caused by an air bubble in the capillary. Take a look at this page https://www.nucleics.com/DNA_sequencing_support/DNA-sequencing-sharp-spikes.html
If you send me the trace I will take a look at it and let you know.
Cheers
Daniel Tillett
Nucleics SupportThe primers really should be closer in Tm than what you have. I suggest that you try making your antisense primer shorter to bring down it’s Tm.
Daniel Tillett
Nucleics SupportHi Mira
If your trace is giving “weak or failed” traces at the end it means that the signal at the end of the trace is too low to be improved by LongTrace. I can’t really tell you much more without seeing you trace, but if you open you trace in a trace viewing program like FinchTV you can have a look at the signal at the end of the trace. You need to look at the raw data as this is what LongTrace uses to do the basecalling. If the peak signal at the end of the trace is very weak (basically flat) then LongTrace is not able to do much to improve the basecall.
A trace file is just the ab1 file that contains the sequence data peaks. I don’t really know why they are called trace files – I guess there is some sort of historical reason for it.
The free service has a limit of 10 files per day – it is really only for tiny users and people wanting to try LongTrace. If you want to process more files then this you need to register for LongTrace FTP.
Finally, have you tried PeakTrace too? Sometimes it is able to improve traces that can’t be improved by LongTrace. The link for the free PeakTrace version is here:
Cheers
Daniel Tillett
Nucleics SupportWe recommend that ABI 3700 users use PeakTrace rather than LongTrace as PeakTrace not only offers better basecalling but supports a wide rang of chemistries and POP types.
Daniel Tillett
Nucleics SupportWe have place the help manual for the LongTrace:Box system online here:
Daniel Tillett
Nucleics SupportI should now add that we know recommend using our PeakTrace software as it has much better supports for the ABI 3700 sequencer than LongTrace. It also supports the really old ABI sequencers like the ABI310 and ABI377. You can find out more about the PeakTrace basecaller is you are interest in try it.
Daniel Tillett
Nucleics SupportHi Resnanti
How LongTrace works is a trade secret, but in general terms it does the same type of processing that ABI’s software does only we are better at it than ABI. I know these seems strange as the guys at ABI are pretty smart. I suspect the ABI management doesn’t think it is a priority to improve the processing of their own traces and hence the software people there don’t work on it.
In the end it doesn’t really matter – what matters is does LongTrace improve your own traces and the best of finding that out is trying our free LongTrace DNA sequencing service. Hopefully you will like what you see 🙂
You might also want to check out our new basecaller PeakTrace. This works much like LongTrace, but on a wider range of machines (it can even improve ABI 377 trace). You can also try it for free at the PeakTrace DNA sequencing basecaller page.
Daniel Tillett
Nucleics SupportHi Ron
That would be great. I will email you directly about this as I really would like to add BigDye 1.1 support to PeakTrace.
Daniel Tillett
Nucleics SupportWe have certainly heard of phred, however, phred is just a basecaller and doesn’t offer the same readlengths as either KB or LongTrace. In rather good news we have developed a new basecaller (PeakTrace) that combines the advantages of LongTrace and avoids the need to use KB. We have written PeakTrace so that it can be easily ported to Linux. So far we haven’t had any paying demand for a Linux version, but hopefully in the future this will allow us to get away from Windows 🙂
Daniel Tillett
Nucleics SupportIf you are interested in using LongTrace for the 3700 we suggest you check out our PeakTrace service as it performs much better on 3700 traces than LongTrace does.
Daniel Tillett
Nucleics SupportHi Anonymous
I hope we are not insulting our customers by explaining why we don’t just sell the software. The LongTrace software is computationally complex (it takes 7 to 15 seconds to process each trace). The mac mini is a surprisingly powerful computer (about equivalent to 3Ghz Pentium 4). To run LongTrace in a reasonable time it needs to be run on a fairly powerful computer – something that the mac mini offers in a small and silent package.
The major problem we face with just selling the software is that LongTrace uses the KB basecaller. LongTrace just process the traces and they are basecalled by ABI’s KB basecaller. Because of the difficulty of setting this up correctly (we need to use KB in a non-standard way) it makes for a very brittle system, where unless we know exactly the state of the system it doesn’t work. Given that the extra cost that the mac mini imposes on our customers is very low (~5% of the final price), and the fact that we are then faced with supporting only one system, we think that this makes sense for our customers – I would hope they value having a reliable, working and supportable system even if it comes at a slightly higher price.
Daniel Tillett
Nucleics SupportWe have decided to move support for the 310 to our new software basecalling software PeakTrace. PeakTrace offers all the benefits of LongTrace, but with PeakTrace we are able to support a wider ranger of 310 run conditions. For more information please visit the PeakTrace information page.
Daniel Tillett
Nucleics SupportYes you can. You don’t need a robot to set up dLUTE SEQ as you can also do it by hand if you want.
Daniel Tillett
Nucleics SupportWe have finished developing the dLUTE SEQ CF formulation. We have been able to keep the dLUTE component the same as the conventional dLUTE SEQ reagent and achieve the change by just modifying the phase indicator solution. Consequently, dLUTE SEQ CF will be priced the same as the standard dLUTE SEQ reagent.
We should have a description and ordering details up on our web site in the next few days, however, if you wish to order it now just order the standard dLUTE SEQ reagent and make a note that you wish to receive the CF version.
Daniel Tillett
Nucleics SupportHi Andrew
The reason is basically the cost of the software and hardware components of the LongTrace:Box system means that we wouldn’t make any money if we charged less per month 🙂 You could just used the 5000 trace per month system and not worry that you are not using all the processings per month. This will have the effect of increasing the average cost per trace a little, but this should not be excessive – the price per trace would move from 10 cents to 16.6 cents a trace.
At 3000 traces a month you might find that it is better to use our online service instead – not only would it be cheaper (less than 12 cents a trace if you by a few month in advance), but it comes with the performance guarantee.
Cheers
Daniel
Daniel Tillett
Nucleics SupportHi Andrew
No this is not the only facilities using PeakTrace, just those that are happy for us to mention publicly that are using PeakTrace. The total number of sequencing facilities and companies using PeakTrace is currently more than 50. Unfortunately for us many companies don’t want their competitors to find out about PeakTrace (they want to use it for themselves only) so they don’t let us publicise that they are using PeakTrace.
Cheers
Daniel Tillett
Nucleics SupportHi Rob
This is hard to answer as the 3700 traces don’t have quality scores it you can’t really measure the read length improvement. This release more improves the number of traces that are successfully processed rather than the amount that any one trace is improved. I should say that there is still quite a bit of work to do in regards supporting the 3700 so you should regard the current version as just a beginning.
Cheers
Daniel Tillett
Nucleics SupportThis problem (ie dye peaks) may still be caused by a clean up problem even thought it doesn’t seem possible at first. The reason for this is the exosap method does not remove salt from the reaction (in particular MgCl2). If you clean up the reaction using ethanol precipitation then the extra salt present from the exosap will cause the leftover dye to precipitate.
The solution to this problem is to reduce the amount (or concentration) of ethanol used in the clean-up. Try adjusting by a few percent at first because it is easy to go too far and lose the pellet.
Daniel
Daniel Tillett
Nucleics SupportYou most certainly can. The traces that QualTrace marks as “bad” are exactly the same traces that are not improved by LongTrace. If you only upload the traces that QualTrace things are “good” then not only will it be quick to upload, but the processing will be quicker too since the server doesn’t need to waste time processing traces that won’t be improved.
Daniel
Daniel Tillett
Nucleics SupportNo there is no linux version of the LongTrace:Box system – I wish there was as I am rather sick of Windows. The reason for this is there is no KB base caller for linux – this means that while we can process the traces on linux, we can’t base call them. I don’t think many people would like a trace file without a base call!
As for why we used an Apple Mac Mini three reasons: has a powerful CPU, it is small, and silent – the price is pretty good too 🙂
Daniel
Daniel Tillett
Nucleics SupportWe have now released this version to the LongTrace service so you can now try LongTrace processing on 3700 traces via the LongTrace FTP service or the free LongTrace webservice.
Daniel Tillett
Nucleics SupportOne piece of interesting news is we have been able to develop a version of LongTrace that works with the ABI 3700. This means that now there is really no good reason to use CounterTrace.
Daniel Tillett
Nucleics SupportYes cooling is one of the hardest things for the PCR peltier unit to do. If you you can one additional tip is to place the PCR machine is a well cooled room with good ventilation to help make the cooling step less of a strain.
In this light I once used to run an air cooled capillary PCR machine in a 4˚C cold room – it really helped speed up the thermocycling stage.
Daniel Tillett
Nucleics SupportWith the release of our free standing LongTrace version (LongTrace:Box) there is even less reason to use CounterTrace II. With LongTrace:Box you get all the benefits of CounterTrace II without the need to use the CounterTrace standard.
Daniel Tillett
Nucleics SupportThe cost of the standalone version is quite a bit cheaper than the online version at $500 for 5000 trace processing per month (10 cents a trace), $750 for 10,000 trace processing (7.5 cents), and $1250 for 25,000 traces processing a month (5 cents).
The downside of the LongTrace:Box system it doesn’t come with any performance guarantees (ie you are charged per trace processed not per traces successfully processed) so you have to be processing a minimum number of traces per month for it to be cheaper (around 2000 successfully traces per month).
Daniel Tillett
Nucleics Support
