32 replies to this topic

[hobglobin]

But 4 ng/microlitre is less than 0.5 ug/50 ul (= 0.01 ug/microlitre = 10 ng/microlitre).
Anyway I'd go with DNA as absolute value that should be in the mastermix that is 10-100 ng genomic DNA, and don't calculate the final concentration.

Edited by hobglobin, 24 February 2013 - 11:07 AM.

[vanu]

yes I know that, but if I want to add DNA at 10 ng/ul, I need the stock to be at 250ng/ul, and as I cant extract that large concentration, I was asking if there is a way to increase my stock concentration by making different extractions

[hobglobin]

vanu, on 24 February 2013 - 11:12 AM, said:

yes I know that, but if I want to add DNA at 10 ng/ul, I need the stock to be at 250ng/ul, and as I cant extract that large concentration, I was asking if there is a way to increase my stock concentration by making different extractions

okay then you have 500 ng in a 50 microlitre reaction which is far beyond any recommendation, anyway for this you can use columns (kit) or make an ethanol precipitation and resuspend the DNA in a smaller volume finally

[GNANA]

see vanu dnt get confuse with concentration and obsolute value. let us just go with wat your pfu protocol says, as u mentioned they say a maximum of .5microgram/50 microlitres (obviously this s far beyond any recomendation as hodglobin said above), which means you will be having  500ng DNA in a PCR reaction. even if you want to use the maximum conc as your protocol says, your template stock having now is enough concentrated and no need to concentrate it further, that is your conc now is 100ng/microlitre, in which if you add 5 microlitres, the total DNA you have added now is 500ng or 0.5 microgram/50 microlitres or in your words it is 10ng/microlitre (for 50 microlitre PCR reaction). but this is way too much,
so if you do PCR with a TOTAL of 30 ng and above upto 100 ng/reaction or even 200ng/reaction, you got to add 0.3 microlitres (30ng) to 2 microlitres (200ng) from your current stock (100ng/microlitre).

[phage434]

Fecal material often (always?) has strong PCR inhibitors. I'd suggest the opposite of most of these suggestions -- that you dilute your sample, perhaps 100x. I'd also strongly recommend that you attempt to find a positive control of some sort to check your primers and verify the appropriate annealing temperatures. Are you using a master mix? I'd suggest one. While mixing can provide more flexibility, that is often a problem not a feature, especially if you have no amplification, as in this example. If you continue to have problems, please report the exact primers, intended template, enzymes, and cycling conditions you are using and we can perhaps help more.

[vanu]

Ok, now I have completely understood, thank you very much for the great explanation, really.Now i could try it again and hopefully will find out a clear band!thank you so much

[vanu]

Dear Phage 434,

Yes I´m still having problem, as I have added up to 400 ng of DNA (extracted from faeces by QUIAamp DNA stool mini kit) to the reaction and there has been no amplification. I´m quite surprised as I have already analyzed these same samples by RT-PCR with SYBR Green and there seems to be amplification and the standard curves were good, I mean we obtained a unique peak for each analyzed bacterial group. As now I´m performing conventional PCR with pfu polimerase and other type of primers suggested by the enterprise in order to obtain the appropiate PCR product for posterior next generation sequencing, we are starting to think that there could be a problem with the primers...

I have 28 pair of primers so I will give you an example of one of the pairs:
16S-0515F 5’-TGYCAGCMGCCGCGGTA-3’
16S-1061R 5’-TCACGRCACGAGCTGACG-3’
~560bp V4-V6 region, GIT
For each of these sequences I should add a MID as tailing, so the final primers used are:
F: TACTCTCGTGTGYCAGCMGCCGCGGTA
R: TACTCTCGTGTCACGRCACGAGCTGACG

On the other hand, I use pfu polymerase from Promega and the tested conditions are:

Hot Start at 95ºC
1. Initial Desnaturation: 95ºC, 1 min, 1 cycle
2. Desnaturation: 95ºC, 0.5 min
3. Annealing, a gradient temperature test from 60-50 degrees (but I have also tried lower temperatures), 1 min
4. Extension, 74ºC, 4 min
(the last 3 steps, 40 cycles)
5. Final Extension, 74ºC, 5 min, 1 cycle
6. Soak, 4ºC, Indefinite, 1 cycle

I would really appreciate any help/suggestion,

Thank you very much

[Trof]

You are using 200ng in your 50ul reaction, the protocol says there should be no more than 500ng in 50 ul, so it's OK.
Other thing is that has to be considered in case of fecal samples is there may be inhibitors precent. So actually lowering concentration of DNA may give better results or you may try some PCR aditives that reduce inhibition, like BSA if your reaction isn't running.

For start it could be helpfull to 1) know the PCR product length that's for sure 2) optimize the reaction on DNA that has been proven to amplify in other assays (so free of inhibitors), it doesn't need to be from the same source, just something that contains the region you are amplifying.

[vanu]

Thanks.
I´m expectin a product lenght of 560bp. I have not use BSA to perform the conventional PCR,but I used it for the RT-PCR, so the failure for amplification might be due to PCR inhibitors...?

[Trof]

Unless you optimize new assay on a verified DNA, you can never rule out the inhibition, especially from a fecal sample.

[vanu]

Hi,

I just wanted to tell you that I have finally obtained my band of interest!So thank you all!
But now, that I have solved my problem with PCR reaction, another problem has come to me...even if I could perfectlly observed by band, there are also some inespecifities so, in order to purify the PCR product for posterior sequencing, I should do it performing a band purification from agarose gel, However I have already try it several times and have only obtained DNA concentrations ranging 10- 20 ng/ul, can anyone help me with this?

PD: I use QUIaquick Gel Extraction Kit, and have made a 0.8% agarose gel, also, I have changed a little bit the instructions indicated by the suppliers, as heating till 60 degrees the elution buffer...but nothing has improved the yield. Any suggestions?

Thank you very much in advance

[Trof]

10ng * 30ul is 300ng, which counting as 80 % recovery of  375 ng is expectable for a PCR band that is not extremely bright.

You can use Qiagen MinElute gen Extraction columns, that have elution volume reduced to 10ul. Overal yield will be the same but higher concentration.

For sequencing generaly 300ng is more than enough, but problem is wih concentration about 10 ng, the spectrophotometrical measurement is too inaccurate. Concentrated eluate helps with this.

[vanu]

But for sequencing they are asking me a minimun quantity of 1.5 ug and 20 ul...
so even if I increase a little bit more the concentration it wouldn´t be enough. What about if I perform a PCR of the PCR product?

[hobglobin]

Three ideas: You could pool the PCR products of several reactions (no idea if this is a good idea, but with a good taq and reliable primers it should be possible), try to optimise the PCR reaction to get more efficiency and therefore product you want and less unspecific products (no idea if this works with your primers and templates)
Third option: Cloning and let bacteria do the job and then you send a purified plasmid plus insert for sequencing

[Trof]

Are you sure they want a one and half of microgram? For sequencing a 560bp product?  What sequencer are they using? For a single reaction with product of such length on ABI Prism 3100 I need 10-15 ng of template in reaction (volume can be up to 6 ul).
I have absolutely no idea what would anyone do with a microgram of DNA in sequencing reaction..  

There is AFAIK no way to get microgram of  ~500bp product unless you pool multiple reactions and precipitate to decrease volume.

Cloning every product is a pain in the ass and they would probably require even more micrograms, because plasmid sequencing requires higher amount of template in reaction in contrast to smaller products (around 100-200 ng).

I would ask them if they really require 20ul of  75ng/ul for a product this short or find more flexible sequencing service.