I would be little worried seeing only smears in the DNA isolates. Did you try to overexpose the gel, maybe there is a very faint genomic band? Maybe another protocol would work? There are few protocols optimized for isolation of DNA from phenolics rich materials (e.g. http://www.academicjournals.org/article/article1380723939_Maltas%20et%20al.pdf ). Nanodrop reading does not give you idea about DNA integrity, it's just the concentration measurement.

Hey Joanna,

Thanks for replying. I know exactly what you are saying, that is why I am trying to troubleshoot my extraction protocol as well. But I am curious because these smeared DNA do give amplification in say like 50% of the reactions. I prefer to ignore the nanodrop readings and only do that for checking the purity basically. 

Also, as the case maybe, there could be some phenolics which might be interfering with my PCR. I have read that BSA should check them, but I need a conformation on the same, or any other/better additive which may sequester the phenolics while I carry out the PCR.

Thanks for leading me to such protocols, I'll try and see which one works best.

try this procedure

I wonder how much of the template you're adding to the PCR reaction? Diluting it may help. Best of luck and let us know if any of the additives helped!

Depending on which protocol you are using, you need to regularly titrate the method, especially when it starts to play up. Usually it is the magnesium chloride salt volume that needs to be altered. You may titrate with slightly different quantities of DNA as well. Hope that this helps. PCRs just stop working sometimes and why a different volume of one of the ingredients suddenly gets it to work again is a mystery but a quick titration is often the answer..same goes for temperature. There are some excellent biomedical trouble shooting websites that give great advice. Google 'troubleshooting PCR'!

@Joanna. I really think diluting these samples might serve the purpose. I am planning to try out everything I can to troubleshoot this PCR thing, so I am definitely going to give it a try. As a matter of fact, 1:10 dilutions have worked well for similar PCRs, although the DNA was extracted from fresher tissue.

Also If you find time to check out the images in my next post, please opine.

@Artur I am attaching the gel images I got for nascent DNA, and also certain PCR gel check images. Please see to them when you get a chance and suggest me something. Thanks for your help.

Also, I am attaching the protocol for your reference.

Dear Annwyne,

Thanks for replying. If you have some time and happen to go through my gel images, please suggest me what you think might be wrong with my PCR? I am definitely going to give a try to increased Mg2+ concentration. Also, you think I should use BSA as it can help me with the phenolics?

In the top gel, the smearing does indeed indicate that the DNA is degraded, so nuclease contamination must be avoided. Another cause of smearing may be too high a voltage for electrophoresis.

If too much salt is in the DNA smearing can occur and an ethanol precipitation could remove these. Protein contamination can also have this effect and a phenol extraction could remove the protein before electrophoresis. I have only worked with human samples and am not too sure about trees.

Did you quantitate the DNA using a spectrophotometer?

In the second gel, the products are very faint which may indicate an insufficient quantity of DNA loaded in the gel so you could increase this amount not exceeding 50ng per band. Another possibility is that the gel was allowed to run for too long and the bands ran off the end. Alternatively, the gel was of too low a concentration and products ran too quickly or the current was too high. The wavelength of light used to visualise the bands may wrong if it is a ethidium bromide gel and check the appropriate wavelength for your particular gel.

In the third gel down, it looks as though the calibration band runs to too high a value and the gel should be run for a longer time to allow your samples to run further in the gel. Could the current be too low or gel too thick? What calibration did you use?

BSA is thought to help with phenolic contamination. For human DNA, I used a chloroform extraction method.

This paper may help-

Plant Molecular Biology Reporter

March 2006, Volume 24, Issue 1, pp 81-91

A rapid method for tissue collection and high-throughput isolation of genomic DNA from mature trees

Josquin F. G. Tibbits, Luke J. McManus, Antanas V. Spokevicius, Gerd Bossinger

Sorry not an expert in plants but hope this helps.

Thank you, DR. Houldsworth for analyzing the gels and letting me know what could exactly be wrong. I think indeed these gels suffer from one or the other flaws you've mentioned.

It is my first time dealing with plant samples too. I find human samples far too easy than plants, especially woody ones.

Thank you for your help.

Thank you Dr. Burzynski for your reply. In addition to this protocol I have used, I am also using Qiagen columns for Isolation, however I got very less quantity of DNA as per nanospec analysis.

These are up to 5 year old herbarium samples. I am trying to amplify the barcode psbA_trnH (~700-800bp amplicon) and I used the ladder accordingly (100bp), however, the fragment seems be an artifact or contamination.

Hi Raunaq, from your gels there seem to be only one well showing (number 17, 3rd gel, lower row) a faint band with your product (although it's hard to tell the amplicon size for sure as the 100 bp ladder hasn't run long enough). It could be an artifact of course. I don't really find qiagen columns great for genomic DNA isolation in terms of yields, but they work good for some people. Good old CTAB usually works the best in my hands. I haven't realized before that you're trying to isolate gDNA from herbarium specimens. In such case there seems to be plenty of good ideas included in the following paper: https://www.researchgate.net/publication/259700673_DNA_extraction_from_herbarium_specimens

I can only recommend being very, very gentle while extracting genomic DNA. Best of luck!

Article DNA Extraction from Herbarium Specimens

Hey Joanna,

Thanks for providing me this pdf. I shall go through this and see what comes around. Right now, I am using one of the CTAB protocols for fresh isolation. I shall subject it to PCR and see how it turns up.

Thanks for your help.

(I am attaching the publication from which I am borrowing the protocol)

Dr. Burzynski,

I wrote this question for two particular reason, which I think weren't clear in my mess. 

a. These are herbarium specimens, up to 5 years old.

b. These are tree specimens, highly rich in polysachharides and phenols.

I have used both, classical (non CTAB) and column based methods initially and they both have worked fine in my earlier PCRs on different samples. However, a and b applies here, which I think are the reasons for no amplification.

I mentioned that I have used a 100bp ladder which is very much efficient for estimating an amplicon of ~700-800 bp, which as the case is in here, is my expected size (which in fact I made clear in my post, and I thought it was obvious that I am indeed referring to the fact that whatever is amplified seems to be an artifact to me). HOWEVER, the fragments amplified are way smaller than expected. I have a clumsy gel image because I was just looking if it has amplified any fragment of approximate size or not. If it had, I'd have processed it further for sequencing, which is what I really intend to do.

I did not make a really long question because rarely anyone goes through such long questions; people tend to skip them.

I apologize for wasting your time on this petty question of mine and messaging you personally for help. No offence taken, but you could have just ignored this question when it was unable to meet your high standards.

Thank you.

Dear Raunaq,

Please don't be disheartened. Your question and the responses will be helpful to many researchers with similar issues and a very valuable exercise indeed. It's when we are not afraid to question that we gain the most. This vehicle for international networking is a huge asset and privilege for all of us so let's celebrate it. Best of luck with all your ventures!

Annwyne

Raunaq, check out the attached paper for a possible additive to try for this...

Thank you, Dr. Houldsworth for your support.

Thanks, Dr. Gallup for attaching a paper for my reference.