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The updates are continuing – we have finished all the USA ones, Australian, NZ, most of the European and have make a good start on the Asian facilities. It is certainly a massive job!
We have continued to update the reviews – another new bunch have been posted over the last few weeks.
Anything with a lot of humic acids in it hard to do. My advice for those reading this question is use the protocol I published in collaboration with Ed Philips lab.
Improved methods for the isolation of cyanobacterial dna from environmental samples
https://www.researchgate.net/publication/227680979_Improved_methods_for_the_isolation_of_cyanobacterial_dna_from_environmental_samplesThe best method is to use 10mM Tris / 0.1 mM EDTA (pH8.0). Let sit at room temperature for 20 min and then vortex lightly. You can do it on ice if you let it sit longer. The key is to let the DNA rehydrate before trying to vortex.
One tip to watch for with genome DNA with PCR is make sure the DNA is not too high molecular weight (~20kB is best for most applications). If the DNA is too long then it can be hard to pipette consistently. You can shear the DNA by pipetting up and down a few times.
This is really an impossible question to answer as there are so many variables. The 3130 is certainly the better sequencer, but it of course much more expensive.
Daniel Tillett
Nucleics SupportThe simplest way is just to blast your sequence at the NCBI site. If your sequence isn’t in GenBank (unlikely these days) then you can always compare to your known sequence using the blast 2 sequence option.
Daniel Tillett
Nucleics SupportIf you want some feedback on your traces I can have a look at them if you write to me directly.
Daniel Tillett
Nucleics SupportYou can get secondary products in the PCR templates. Normally they are at a low level and so you can’t notice them, but when main signal ends they come through as weak peaks that the KB basecaller then calls.
Daniel Tillett
Nucleics SupportThe details on how to purchase PeakTrace are on the PeakTrace page.
Daniel Tillett
Nucleics SupportFirstly, Taq is not unstable at room temperature.
Secondly, I am not sure where you got this 1:100 template to primer ratio from but this is also wrong. I guess if you your assumptions are wrong then you should not be surprised when the results don’t make much sense 🙂
Daniel
Daniel Tillett
Nucleics SupportDo you have a positive control for the reverse transcription step? Do you have another PCR primer set that you can use on the cDNA (a house keeping gene) that you can use to check that cDNA is OK. Have you tried spiking in DNA to make sure there are no inhibitors present?
Daniel Tillett
Nucleics SupportThis is lab forklore, but I can say I have tested it myself and it true.
Daniel Tillett
Nucleics SupportHi Kate
In the fridge short term and in the freezer long term. It is not a good idea to store PCR products if you plan to TA clone – storage seems to remove the 3′ A overhang.
Daniel Tillett
Nucleics SupportThis sounds like you have a SNP in your sequence. Since you are sequencing from PCR product you actually have two different PCR products from each allele. If one has a small insertion or deletions in one of the alleles then the sequencing will go out of phase after the point of the SNP.
Daniel Tillett
Nucleics SupportIf you want to make a 54 base oligo just contact a company that makes them and give them the sequence you want. They will make it and ship it to you.
Daniel Tillett
Nucleics SupportThe problem with using such low binding temperature primers is two fold. the first is Taq does not have much activity at 30C. The second is the template will have a fair amount of secondary structure that prevents the primer binding. The experiments we have done developing UniSeq showed that you really want to stay above 37C annealing temp and also have at least 11 bases binding.
Daniel Tillett
Nucleics SupportI would say this is due to the secondary of your template. One of things people forget is in bigdye there is no GTP but ITP (inosine). The polymerase actually doesn’t like ITP and when faced with significant secondary structure sometime other nucleotides are inserted.
Daniel Tillett
Nucleics SupportIs there any reason why you can’t use the same master mix for both DNA’s. If the only thing that is different between the two mixes is the DNA then keep them the same. Use the human DNA conditions for your plasmid control. If the conditions work for the more difficult human DNA templates then they will work for the much easier plasmid.
Daniel Tillett
Nucleics SupportYou will need to provide more information to get an answer – what kit?
Daniel Tillett
Nucleics SupportThe problem with storing PCR products in water is if you have any nucleases around. It is better to store them in 10/0.1 TE. This way the pH won’t drop from absorbance of CO2 and the EDTA will keep the nucleases from chewing up the DNA.
Daniel Tillett
Nucleics SupportNo, but you can lease a computer with it on it so you can run as a standalone system at your facility
Daniel Tillett
Nucleics SupportYes it might be methylation that is the cause. Unless there is another enzyme that is not affected by methylation that you can use you won’t be much you can do about this. Might be worth trying another enzyme site.
Daniel Tillett
Nucleics Support
