Hello again. after read all answer i give it a go to PCR this product. im little bit pesimist but the result is shocking for me :). but im still curious why in genom DNA there is no band at all. but in PCR product that very clear band. im using ITS4 and ITS5 for primer because is a fungi. 1.5% agarose, 1x TBE and 50 Volt.

Can be eg. humic acids. Are the samples brownish?

Dear Fuad Dwi Atmaja

Please go for these steps:

1.check your spectrophotometer with another DNA observed its band on agarose gel.

2. If it's Ok, load it again Two-fold, three-fold, etc.,

3. perform PCR with the DNA (4-5 micro liter DNA)

4. let me look at your gel picture.

Dear Fuad Dwi Atmaja

1. Your DNA purity is not good ! you have some RNA pollution in your final product.

The best is 1.8 - 1.9 max 2

2. As Miss Ravanbakhsh mentioned, check andother DNA observant with your Spect.

If It was Ok, chenge your electrophoresis tank, and run it again.

But if it was not:

3. Check your extraction process, and fill RNase adding in your process.

Regards.

Was there phenol during extraction or just chloroform. Phenol:chloroform method may give strong background signal if not washed well.

To purify genomic DNA (after extraction), one may use isopropanol (2 volumes)-> shake well-> DNA is visible-> centrifuge, wash with 70% ethanol

Free nucleotides or anything else similar to purine, pyrimidine, tryptophan will give strong signal as well

I think you might have poly-saccharides still associated with your DNA, effectively changing its charge. If that is the case your DNA will move opposite to the normal clean DNA. try to switch the + and - on your electrophoresis and see if that is the case. You will need to use some Lyticase or similar enzyme to get rid of the polysaccharides. You still can use your DNA for PCR provided that you put enough BSA.

my2c

Dear alentyn Oksenych

I think Fuad Dwi Atmaja should do PCR with it, if it didn't work, OD=2 could be any thing other than DNA.

Check also the 260/230 that gives you the relative content of inhibitors (eg, phenol-like compounds). Use Nanodrop to get both 260/280 and 260/230 values and have a look at get the absorpance profile (~220nm to 300 nm) of the extract. For purified DNA，you should get a clear peak at 260 and the 260/230 is good (~ >1.5) and 260/280 >2.0.

@Krzysztof Pudelko : no sir, the sample are bright clear,

@Reyhaneh Ravanbakhsh :

1. Already done that, i already checked using different DNA and the result is good in both spectro and elfo

2 and 3 okay i will do that

4. Uploading file name "sam_1374"

@Davood Zaeifi

1. how can be sir? in my litterature 1.8-2.2 is good purity :D

2. already done that sir

3. yes i already using RNAse in my buffer

@Valentyn Oksenych no i wasnt using phenol because i know is hard to remove the phenol, so i only use CLAA 24:1, i had use propanol but give me a bad result though

@Petre Ianakiev yes yes i think the polysacharide is the problem here, i never think to reverse + and -, how it can be done? i dont know to reverse

@daniel deng okay ill give the ration 260/280 and 260/230 when im going to my lab :D

Turn the gel 180 degree....also if the polysaccharides are the problem, after precipitation of DNA you will see gel like pellet, wash that with 50% EtOH (not 70%)

Also your gel runs strange, check the pH and salt concentration of the buffer.

@daniel deng im already in my lab this is the ratio-

A230: 0.214; A260:0.244; A280: 0.161; A320: 0.081

260/280: 2.037; 260/230: 1.226

@Petre lanakiev yes yes i have get like pellet. what is that? i wash using ethanol 70%. so i must wash using ethanol 50%? is that significantly different 50 and 70%? (just curious) . ph in buffer is not wrong and gel concentration 1%.

Fuad,

Based on the raw spectrophotometric data above, how have you calculated your 260/280 ratio? I get a value of 1.51.

Nanodrop can seriously overestimate DNA concentration (which might be consistent with your gel image). Have you got access to a fluorometer?

Petre's idea of running the gel sideways is neat idea. Worth giving a go for sure.

If you are confident that the DNA is actually there, you could always try doing a PCR and seeing what happens. Make sure you have a robust positive control (good primer set you know that works, and maybe another DNA sample you have done the PCR on before that also worked), and I would do a serial 10-fold dilution series of your sample so that if there are any inhibitors, they will get diluted out. Often with PCR, less DNA is better than more, especially if there is inhibition to do with the sample.

Let us all know how you get along.

Steve

This is a common problem in DNA extraction from plant. Spectrophotometer can read nucleotides or degraded DNA but it does not mean you have a good DNA quality.

Your problem is in one or more of the following:

1- You are using an old CITAB as a detergent. Solution: Use a fresh reagent.

2- Old phenol/chlorophorm. Solution: Use a fresh reagent stored at 4C.

3- You are using an old tissue, tissues which already tern to senescence stage (apoptosis). Use young leaves or whatever tissue.

4- You did not precipitate DNA using the correct concentration or temperature. Solution: Use a fresh isopropanole or ethanol 100%.

Based on my experience, you may get some PCR products out of this DNA however, the results may not reliable.

Good Luck

Mack

It is not necessary spectrophotometer meter will give u pure DNA, it can b free nucleotide .you must check purity by other method like qubit, bioanalyzer, and PCR. OD of DNA sample must be in range of 1.8-1.9 for good quality. 2.1 OD is showing washing was not good.

Yes i agree for dna quantification go for gel , qubit sometimes od/spectrophotometer is not reliable

Dear alentyn Oksenych

As Mahmoud Yaish mentioned , Extract another DNA from fresh leaves or tissues with Yaish's protocol. Try to do this protocol carefully.

good luck

If you have Qiagen gel extraction kit - that might help with polysaccahrides. It breaks down the polysaccharides to smaller pieces that is less of a problem. It will give you bad spectrophotometric reading, use pico- green instead .

Hello again. after read all answer i give it a go to PCR this product. im little bit pesimist but the result is shocking for me :). but im still curious why in genom DNA there is no band at all. but in PCR product that very clear band. im using ITS4 and ITS5 for primer because is a fungi. 1.5% agarose, 1x TBE and 50 Volt.

Nice band!

I don't know how big the chromosomes of your fungi, but for mouse genomic DNA one does not expect to see band after DNA purification because genomic DNA is very long and will not run in 1%agarose. At the same time, for Southern Blot we pre-digest genomic DNA (about 5-40 micrograms per sample) with relatively widespread enzymes as EcoRV, EcoRI, HindIII etc. After such digestion (usually overnight at 37C) one detects shmear as DNA is chopped to smaller pieces that have a chance to run in agarose

440 microgram/ml means 440nanogram/microliter. If you have used 5 microliters it means 2.2 microgram. I would increase amount of DNA 10folds (50microliters, 22 microgram), digest with 1-3 different enzymes such as EcoRI or HindIII, in total volume of 200 microliters (including5-10 microliters of enzyme, and 10x buffer, 100xBSA or according to enzyme supplier's protocol) 37C for 10-20 hours. Then concentrate digested DNA with vacuum or isopropanol, and get about 50microliter final volume. Run it in agarose gel very slowly in TAE buffer (40-50V) for another 10-15 hours. This is what we do for mouse genomic DNA. Expected DNA sizes several kb

https://en.wikipedia.org/wiki/TAE_buffer

http://www.ajol.info/index.php/ajb/article/view/58763

Influence of DNA treatments on Southern blot hybridization analysis of Fusarium oxysporum F. sp lycopersici and F. sp radicis-lycopersici genomic DNAs

OS Balogun

Abstract

DNA samples obtained by a non-phenol/chloroform isolation method, from three races of Fusarium oxysporum f. sp. lycopersici and f. sp. radicis-lycopersici were treated in different ways with a view to evaluating the effect of three pre-electrophoresis DNA treatments on the outcome of Southern blot hybridization analysis using a Digoxigenin (DIG)-IGS fragment probe. Results showed that where DNA material is scarce, the use of undigested (native) fungal DNA not only saved time but it also gave better hybridization signal than predigestion treatments with EcoRV restriction enzyme. Hot water digestion of DNA prior electrophoresis and hybridization gave the least satisfactory result.

Well done Fuad, and thanks for sharing your result.

Two things you can take away from this. Firstly, may now want to be a bit "pessimistic" about analysing genomic DNA concentration in the way you have as clearly the concentration was out. Secondly, you don't need much DNA at all in a PCR.

This might happen if the Genomic DNA isolated is sheared and the nucleotide may contribute for spectrophotometer detection. Therefore always we need to confirm the DNA on 1% agarose gel. The quality of DNA is found good then estimate at Spect for the quantity.

It happens many a time. See the degree of shearing of the DNA in the solution that you are loading in the Gel.

Iam a victim of this experience, iam working with soil bac DNA, and after extraction and gel running there was no band. I had to increase my DNA on  0.9% agarose at 70v for 120min before I could see distinct bands. Get to know the size of you DNA from literature and see how far you can manipulate the gel running conditions

Try to take reading by Qubit....

Hello every body, i also come across the same problem these days, i extract total genomic DNA from yogurt,on spectrophotometer it show 150 to 220 ug/ml od 260/280 is 1.75 to 1.93 for some sample but when run a gel there is no band at all,although the marker show band,from the above discussion i do not found conclusive results or suggestion that can be used to solve the problem

.

Dear Fuad, Have you find solution of your problem,please share.

Thanks to all, the information you shared save me a lot of time wondering why genomic DNA do not show band during gel electrophoresis. We'll proceed with pcr with our samples although this will be more expensive than a confirmatory test involving genomic dna. Thanks Fuad.