If you'll provide PCR, you can to get product from most your DNA samples successfully. What amount of DNA you used for electrophoresis? What ethidium concetration and agarose concentration you used?

Such picture can be observed requently when you use herbarium material. This may be caused firstly by the fact that all plants has different nucleases, hydrolitic ferments and so on, which can paritially destroy DNA during storing samples. Another way is problems with technique protocol using. Especially grinding of plant tissues in most cases must be provided in liquid nitrogen without any heating and CTAB must be added immediately after grinding. In spite of this fact, please note, that for successful PCR product obtaining you need only few copies of initial DNA. If you want you may to clean your DNA by different kits. But before purifying DNA I recommend to try to run PCR. Also I recommend make sure that smearing bands was caused exactly by DNA, not by polyphenoles, secondary metabolites or polysaccarides or high concentration of EtBr.

I had different problems with DNA isolation from herbarium material too and made some impruvements to standard CTAB techique to increace the quantity of DNA with adequate quality. If you'll interested in you can find this paper on my page here.

Hi, I think the DNA is okay. Try your PCR (gradient PCR; different amounts of DNA). Cheers, Nadine

Is your silica gel working well? Your DNA degradation could due a long time before your sample get dries. I used to perform DNA extraction from silica dried leafs with QIAGEN kit with any problem few years ago. Anyway, with that DNA quality your PCR should work. As Nadine said, try gradient of temperature and differente amounts of DNA. Good luck!

Using column to isolate genomic DNA can exclude DNA fragment, such as QIAGEN kit.

However, as José said, the DNA degradation during drying the leaf can not be ignored.

In addition, add 0.5% PVP to CTAB solution is useful to exclude polyphenols.

If the sample is rich of polysacharides, it is highly recommanded to use column to isolate DNA.

Hi!

though your DNA is degraded a bit it works fine. No worries.

I think the image is inverted. From left hand side 4th well I do not see any DNA, is it blank? Or no DNA in that sample or loading problem? Try to dilute all samples equally (having equal quantity 20 - 50 nano grams per uL by taking little DNA separately) then try PCR it should work fine.

Good Luck!!

Try the KAPA3G PCR kit for your amplifications. I was having trouble with PCR from cambial and leaf tissue, but after purchasing this kit I had much more success. The Polymerase in this kit is engineered to work better in the presence of plant inhibitors.

You may be shearing the DNA, did you vortex the sample vigorously.

If you'll provide PCR, you can to get product from most your DNA samples successfully. What amount of DNA you used for electrophoresis? What ethidium concetration and agarose concentration you used?

Such picture can be observed requently when you use herbarium material. This may be caused firstly by the fact that all plants has different nucleases, hydrolitic ferments and so on, which can paritially destroy DNA during storing samples. Another way is problems with technique protocol using. Especially grinding of plant tissues in most cases must be provided in liquid nitrogen without any heating and CTAB must be added immediately after grinding. In spite of this fact, please note, that for successful PCR product obtaining you need only few copies of initial DNA. If you want you may to clean your DNA by different kits. But before purifying DNA I recommend to try to run PCR. Also I recommend make sure that smearing bands was caused exactly by DNA, not by polyphenoles, secondary metabolites or polysaccarides or high concentration of EtBr.

I had different problems with DNA isolation from herbarium material too and made some impruvements to standard CTAB techique to increace the quantity of DNA with adequate quality. If you'll interested in you can find this paper on my page here.

We did not vortex our sample at all. But since we did not have liquid nitrogen, the time for crushing was elongated hence exposing the DNA to different metabolites (I think). We haven't yet quantified our DNA. We have had similar gel pictures in our previous tries too but PCR showed no results. Hence we were trying different protocols and the above gel picture contains DNA bands of DNA isolated using 6 different protocols of the same sample each protocl modified twice (using propanol or PEG for precipitation). We will be performing quantification and PCR today.

Your DNA sample appears to be sheared and contaminated with other macromolecules. You can use lyophilized samples in place of liquid Nitrogen but the extraction should be faster and with a good grinder or a mortar and pestel. To avoid ply saccharides it is better to keep the plants in the dark overnight and for polypehenol removal addition of PVP or PVPP while grinding the sample with extraction buffer is recommended.

your kit provides treatment with RNA-se?

we did not have RNAse treatment. I quantified the DNA today and a few samples showed A260/A280 of 1.7 while most showed values less than 1.5. and the A260/A230 was considerably low for few of the samples indicating high polysachharide contamination. Is there any alternative for RNAse treatment. I was planning on using pectinase for the removal of polysacharide but haven't been able to find any protocols on it. At what concentration should I use pectinase if i do?

It seems you have degradation of DNA and this is normal for dry leaves since during drying apoptosis will occur during which DNA fragmentation usually happen. One other point, with high phenolics or carbohydrate content you better use make your CITAB buffer 2% CITAB and 1% PVP. The addition of PVP will help to much to clean your DNA from polysacharides and polyphenols

The DNA is degraded to some point at all you samples, except No. 3 and 5, which are OK. No. 13 has impurities and is contaminated (look in the well), but even that kind of samples amplify, so it always depends and you should try PCR and RNAse is not very necessary. Use more efficient polymerase (like Phusion) for PCR and make a few DNA template dilutions - this will help minimize the amount of inhibitors in the DNA sample. Try PCR with your 1) usual amount of DNA template 2) DNA template diluted 2 times 3) diluted 4 times, etc. If your PCR product is quite small (up to 1kb) you should be able to get it, if your product is rather big (2-3 kb) then there is less chance to get it, but you never know. Hope this will help.

I think it's rather the contamination.

hi Ashish

I have attached a paper we published several years ago that describes DNA isolation from plant tissue using a modified CTAB protocol. The protocol is quite detailed and may have some helpful hints for what you are trying to do

good luck

Thank you very much!

Try using a higher grade phenol. Add isoamyl alcohol to chloroform. Make sure chfm is buffer saturated prior to mixing with 49:1 isoamyl alcohol. All mixing (leaf extract with solvents) must be gentle and low temperature. Dna breakdown can be due to higher endonuclease activity in your leaf samples. To prevent endoN activation, use higher conc of Sod. EDTA. DNA extraction Kits are good but in this situation use of generic reagents may be better. Time consuming but resutls will be good! Best of luck....SDR

Hi,

besides trying different concentrations of DNA-template (in particular lower concentrations) you could add bovine serum albumin (BSA) to a final concentration of 3 mg/ml to your PCR master mix. It helps to inhibit toxic effects on your DNA polymerase (Romanowski et al., 1993, AEM59: 3438.)

Hi, I normally do Silica purification (Silica beads with some chaotropic agent, like NaI or even Guanidine). The protocol is very "ancient" so I can't remember the right citation, but there are a lot of variants on line. Elution takes a lot in pure water (1 hour or more, with moderate heating) but the yield is satisfactory and, more importantly, the DNA is stable over a long period. Don't buy any kit, Silica beads are unexpensive.