Dear Dr Norma,

I find in your protocol that after Proteinase K treatment, you are not further purifying the DNA to remove potential PCR inhibitors or Proteinase K (not 100% of the enzyme is expected to get deactivated at 89C), which might be a factor causing poor amplification. I suggest you to purify your Proteinase K treated lysates by Phenol-Chloroform extraction and Ethanol precipitation or simply purify the crude extract using a good quality commercially available tissue DNA extraction kit.

Moreover, considering the PCR with Folmer primers, which are expected to generate an amplicon of ~700 bps, you need to increase the incubation time in your thermal cycling program. Since your gel image is showing some amplicons, I suggest you to repeat PCR with your existing DNA preparation under the following PCR parameters:

95 degree C pre-denaturation (2-3 min), 45 cycles of [denaturation 95 degree C (30 second), annealing 56 degree C (30 second), extension 72 degree C(60 second)] and last extension 72 degree C (15 Minutes).

This may probably improve your results, would like to know if it works,

Best wishes,

SD

Thank you ever so much prof. Datta, I will try that.

Did you measure the dna amount or run some genomic dna on a gel?

It seems to me that your evidence that you have a poor dna extraction is that your pcr failed. It is just as likely that your pcr failed maybe because the primers have degraded or some other issue. Do you have a positive dna control that amplifies to run on each pcr assay so that you can tell if the pcr is working well?. How much dna are you amplifying....it may just be too little or possibly contaminated with pcr inhibitors. Also you are running the gel for far too long. run for about half the time or voltage and the bands will look better if those bands in the lower part of the gel were loaded on the top wells

Dear Paul, I am terribly terribly sorry of not having time to respond you quickly. Yes it is a gDNA, I am trying to do barcoding of my samples.This technique worked well previously (last year), however as suggested I am going to use new primers, it is on the way now from the supplier; positive controls have been done previously, I put only 1 microliter DNA sample when amplifying. I presumed my primers are indeed contaminated, since I kept them from last year usage. How much important to use nuclease-free-H2O instead of ddH2O please? could that also be included as one of the reason?..I found my colleagues got more stable results with marine fish/shark, could that be because fish has less mucus than cephalopods please?. Cheers.

Dear Drs. Sibnarayan Datta & P. Rutland, here is the result following after your suggestions, improved greatly. However, what are those trailing furtherdown the COI bands (marked), please? Thanks a lot.

Hi Norma,

Happy to learn that you have got good results. To me, the COI bands look nice. You can just proceed with purification of the bands and sequencing.

regards,

SD

Probably non specific amplification. Two ways of getting rid of this is 1 Run a temperature gradient of increasing annealing temperature and hopefully as the temperature increases the NS band will disappear and the specific band will remain.

2

ADD increasing amounts 1%, 2%, 3% ….8% Of dmso ( do not go above 10% it kills the reaction ) to your pcr mix which has the same effect as increasing annealing temperature and there may be a concentration of dmso that gives you only specific product.

If both of these fail to clean up your pcr then there is a very similar sequence to yours elsewhere on the genome ( a pseudogene or another member af the gene family) and you may have to redesign your primers or just ignore the large band as it is well separated from the one you want. Sequencing both will probably give you the answer.

You may find that decreasing the annealing time and extension time in your pcr lessens the large band also

Dear Dr. Datta, thank you very much for your guidance..it works!

Dear Dr. Rutland, it happened before, pretty clear secondary bands. During further process in the sequence, (without my presence) they said those secondary bands gave no information at all. However, I would like to go after your suggestion about dmso increment; how much volume should I add please? 1/2 microliter? Have no experience working with that stuff though. Thanks for your kind attention.

Hello Norma,

using dmso run 7 tubes tube 1 no dmso

tube 2 if using 20ul pcr reactions replace 2ul water by 2ul of 10%dmso ( final is 1% dmso)

tube 3 replace 4ul water with 4ul of 10%dmso ( final conc is 2%dmso in tube)

tube 4 replace 6ul water with 6ul 10%dmso

and further concentrations of dmso using 1:5 dmso to replace an equal amount of water to make the higher concentrations 4%-, 5%, 6% , 7% and 8%.

By the way I did not answer your earlier question about using water to dilute oligos. I never would use water as it becomes slightly acidic and depurinates oligos. TE is better as it remains alkaline and the edta stops any contaminating nucleases from degrading your oligo dna

Dear Paul, thank you so much for your detailed elaboration. Practical things not mentioned in the journal is very important if one get stuck.

By the way Paul is that mean that you replace dd-H2O and/or NFW with entirely TE buffer? What about the xoncentration of the buffer? Do we need to dilute it or simply replace the water volume with it, please?

Cheers...

Most primers come dry from the supplier,Just dissolve in 10mM TE for storage then again for daily use in TE or water. I have re read your last question and it is ambiguous. I have been assuming that it is the larger(ringed) band that you want to get rid of while keeping the smaller band, Is this right? If it is the smaller band then my advice will be different

Norma Afiati & Paul Rutland : Yes, there is some confusion regrading the specific band. I considered that the desired band were the ones red circled. Nevertheless, even if the smaller amplicon is your expected product, then I will again suggest you for gel purification of the bands and sequencing (at least a couple of bands can be sequenced at first to check if they are the specific amplicons, you are looking for).

Best wishes,

SD

Truly sorry for not being crystal clear, the ones in the circle is the COI I have been looking for, and my query is about other good quality bands below the COI sir. Sorry..

Ok Forget what I said about getting rid of non specific bands for the moment.It is correct but not relevant. I think that you have a primer dimer small band and the 4 main ways of getting rid of PD are:

1 Use a lot less primer ( cheapest solution) and set up on ice and start cycling immediately

2 Do a hot start pcr by adding enzyme while the mix is already hot ( fiddly but it works)

3 Use a hot start enzyme. (Best solution)

4 Use different primers

What do you think Sibnarayan Datta the small band looks just a little large and is sharper than usual for PD which is often very diffuse but it does look like PD to me?

Also the first 3 samples where the pd is strong have not amplified which often happens when primer is removed by dimerization from the pcr reaction

Thank you so much, I would follow your suggestion one after another to see which one works best to the species I am working with. Is excessive mucous might halt the oligo reaction please? Because the most and always successfull in my case is when I work with Octopus, the least mucous animal amongst the other..thank you.

I do not know about the properties of Mucus which is very complex but if You think that it is a pcr inhibitor and co purifying with your dna then there are a couple of things that might be worth looking at.

1 Try amplifying less dna .....sometimes the effect of having less inhibitor means better amplification so less dna means more product.

2

Try using more Mg in your reaction as Taq needs Mg to work and some inhibitors take Mg out of solution so try doubling the amount of Mg in your reaction with high mucus dna.The worst it can do is make the reaction a bit less specific

3

Take another dna that works well and add some octopus dna to it and pcr. If the pcr fails/works badly then you have inhibition..

Are your od260/280 ratios for other species and octopus both good.?

If everything else fails try a different dna purification method

The 260:280 optical density ranged between 0 75 - 1.56 in all of them. What form of Mg that can be used in the reaction please? Thank you so much for the suggestions..

OD 1.8 would be ideal and I imagine the dna with ratio .75 are the worst amplifiers having much protein or other absorbing non dna material. Most polymerases need 1mM final Mg concentration so usually supplied already in the buffer or mastermix, The type of salt does not matter so make up a 15mM aqueous solution of magnesium chloride or sulphate ( 10x concentration) and if your pcr is 20ul replace 2ul of water with 2ul of the Mg salt solution on a poorly amplifying dna and see if it improves the amplification

I totally agree with you Paul Rutland that the smaller bands are actually PD. In fact, Norma Afiati has also mentioned previoulsy that sequencing of the smaller bands did not yield any information and as you have observed that the smaller bands are strongest where specific amplification is totally absent. This is what exactly happens with PDs.

In my experience, optimizing primer quantity is very helpful in such a case, as you have already mentioned. Also I want to add that often cheaper desalted oligos and older stocks of primers often produce PDs/non-specifics, while HPLC purified primers, although costlier than desalted oligos, give very good specific amplification.

So, in this particular case I would have first tried optimizing different amounts of primer, say from 50 nM to 500 nM, followed by a temperature gradient and if required, a MgCl2 optimization test.

Regarding the issue of mucous, since other DNA specimen are giving good amplification, I think that mucous is mostly removed by the method being presently used by Norma. Even then, if some samples are not showing amplification, other PCR inhibitors have to be ruled out. For that, I would first check those DNA samples on a 1% agarose gel. If HMW DNA is visible, then I would re-purification the samples using a good quality commercial stool/soil/plant DNA kit, since these kits are very efficient in removing a wide array of PCR inhibitors.

I hope this works for Norma Afiati :-)

SD

Even though I also prefer optimizing PCRs to get best possible specific amplification, in practise, many a times nothing appears to work and PDs or non-specifics simply wont go. In those situations, I try to purify the specific bands from gels and go for direct Sangers or cloning-sequencing according to the experiment requirement.

Seeing the gel photo here and good amplification of the COI bands (some are extremely high yield), I strongly believe that the bands can be cut, purified and sequenced successfully. Norma Afiati you can at least try re-running all the 3 bands in the upper lanes and the 1st and 3rd bands in the lower lane, cleanly cut using sterile blades and purify using a good quality gel extraction kit. Then send them for sequencing and see what you get.

Meanwhile if you share you PCR composition and thermal program, may be we can suggest certain specific solutions.

best wishes,

SD