I have a problem in the separation of small DNA by Agarose Gel Electrophoresis (though I optimized with different concentrations of gel). Separation was very poor and always very blurry, smear bands with DNA ladder (every 20 bp scaled up from 20 to 200 bp). I plan to run with SDS-PAGE with thought it will be better, but not sure it will work well. Another problem of using SYBR gold dyes which seems less effectively staining DNA, RNA than using EtBr which we do not want use in our lab. Highly appreciate suggestions.
Hi, Kien. I used silver staining of nucleic acids for detection of SNPs after PCR of alleles. it worked well both for heteroduplexes and for diferenciate secondary structure in sisngle strand DNA. I know that in the "prehistoric ages" of manual sequencing it was used instead of radiactive labelling.No idea if it works with RNA. I don't know were are my notes but in "molecular cloning. A laboratory manual" by Sambrook and Russell it's described a similar protocol (Appendix 9.6):
Rinse the gel in water twice, fix the gel in 10% ethanol (10minutes), remove ethanol and cover the gel in 0.7% nitric acid (6min) remove and pour silver nitrate 0.2% (30min), remove and cover with 125ul of formaldehyde solution (37%) diluted in 100ml of developer (22.9g sodium carbonate per liter) and wait for color development.
all steps in a rocking platform or shaker. To stop the reaction remove the developer and add acetic acid 3%. Good luck
Polyacrylamide gel (denaturing condition with Urea) greatly worked for me to run 23nucleotide ssDNA(same works for RNA). Agarose gels are not preferred to run small DNA fragments/RNA.
I have same experience as you EtBr works equals to 100X times better than SYBR gold. Let me know if you need the protocol for this electrophoresis.
Thanks for your kind sharing! If it is possible I would refer your protocol. Could you please kindly send it to me according to this email.
Denaturing Gradient Gel Electrophoresis (DGGE) is a suitable way for recognizing even 1 nucleotide in a sequence. You can use link below from JOVE:
http://www.jove.com/video/1485/denaturing-urea-polyacrylamide-gel-electrophoresis-urea-page
PolyacryIamide gel is really helpful for separating small RNA and DNA fragments. I have used 15%-20% PolyacryIamide gels for separating 15nt fragments. Also I could see better staining of the gels with SYBR GREEN 2, which has more binding affinity for single stranded DNA/RNA than for double strand. It could be useful, if you would not like to use EtBr.
Separate by electrophoresis in denatured 20% polyacrylamide gel (19:1 acrylamide / bis acrylamide), 1x TBE (tris borate EDTA), 8 M urea). Prior to loading denature sample by heating 95'C with loading buffer with formamide. After running take gel out and stain with ethidium bromide or SYBR safe to visualise DNA.
I have TAE buffer right now. Is it OK if I will not use TBE buffer as your suggestions?
Another thing, I know that Urea is a hydrogen bonding destabilized reagent. Should i put some cares with high concentration of 8 M such as degradation or fragmentation of ssDNA, RNA oligos (we use synthetic ones). Why do I need Urea if I have Formamide in sample preparation?
TAE or TBE ... it depends on your downstream application. TAE is suited for extraction and clonig and can be prepared as a 50fold stock solution. But it has a low buffer capacity, therefore the separation is done at relatively low voltage.
TBE has a higer capacity but the borate interferes DNA extraction and it participates at 5–10fold stock concentrations. You might have a look at TTE (Tris taurine EDTA), which has a high buffer capacity for separtion at higher voltage, is suitable for cloning experiments and can be stored as 20fold stock solution.
I am in complete agerement with Ananda. Poly Acrylamide Gel is the best option for such a short fragment. In fact it is always advisable to go for PAGE in case of such fragments. Some times RFLP studies use the PAGE for better resolution of short fragments. Look for PAGE.
I agree with all the previous comments. A PAGE in denaturing conditions (urea) should be work. They have a high resolution and are ideal for small fragments.
Regarding the staining, if GelStar or other EtBr doesn't work, maybe you could use a silver staining method. it works well for PAGE and the sensibility is higher than the EtBr. If you need a protocol I can look for it in my files.
Hi Miguel, I have not been knowing that silver staining even works to visualize ssDNA, RNA bands. I just thought it works well with staining of protein. I have some protocols for silver staining for proteins but not for DNA, RNA. I am curious to use silver staining so highly appreciated if you could share.
I agree with the previous answers. I get good results by using 20% polyacrylamide gels (37.5:1 acrylamide bis acrylamide), 1x TBE and 8 M Urea. Prior to loading I denature the samples by heating 2´min at 95°C in presence of 5 M urea (final concentration). The gel could be stained with ethidium bromide (I prefer to use SERVA DNA stain G that is also working very well for RNA staining) if you want to use the samples latter. You can also stain the gel with silver but in this case you will be no not able to use the samples for other purpose after that.
Hi, Kien. I used silver staining of nucleic acids for detection of SNPs after PCR of alleles. it worked well both for heteroduplexes and for diferenciate secondary structure in sisngle strand DNA. I know that in the "prehistoric ages" of manual sequencing it was used instead of radiactive labelling.No idea if it works with RNA. I don't know were are my notes but in "molecular cloning. A laboratory manual" by Sambrook and Russell it's described a similar protocol (Appendix 9.6):
Rinse the gel in water twice, fix the gel in 10% ethanol (10minutes), remove ethanol and cover the gel in 0.7% nitric acid (6min) remove and pour silver nitrate 0.2% (30min), remove and cover with 125ul of formaldehyde solution (37%) diluted in 100ml of developer (22.9g sodium carbonate per liter) and wait for color development.
all steps in a rocking platform or shaker. To stop the reaction remove the developer and add acetic acid 3%. Good luck
I appreciate and welcome all comments and suggestions. It is nice to be a part of and to learn from scientific community.
Dear all,
I am facing the same problem as well. Can any of you send me a protocol for analysing 20bp dsDNA ? Many thanks !
I use silver nitrate to visualize SSR PCR product in PAGE but some times it dose'nt work for me and my PCR sampels don't appear well. i can not understand the reason! I use 1.5gr silver nitrate per 1.5liter water
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