- Maria Amaral added an answer:Smear on agarose gel from QIAGEN extracted plant samplesWould anyone know if it is common to observe these types of smears on an agarose gel after extracting DNA using a QIAGEN DNeasy 96 kit and running 500 ng of DNA in a 1% gel at 70 volts for 3 hours inside a 4 Celsius fridge (also tried at room temperature)?
I'm planning on sending these samples for Genotyping-by-Sequencing but the requirements for such procedure include no smearing on agarose gel neither discrete low molecular weight bands, which I think I have. Any input will be appreciated.
I have just joined this Rgate because of the problem you share here.
I am working with DNA from a variety of tropical plant species and for some, I get gel pictures exactly like yours. The smear may depend on some DNA degradation that has already occurred in your samples, or that is happening during extraction. What kind of samples did you worked with?
For the bands, I search on the net and I got 2 possible answer, so far, for this: lipopolysacharides with bind to ethidium bromide (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC208550/ ), and / or DNA fragmentation. (file:///C:/Users/Joao/Downloads/JCT20110500005_97044297.pdf ).
I tried to isolate the high molecular band fraction from gels, but this will made me loose too much DNA. I may just assume the risk and submit the samples as are, but I would help me a lot to know what decisions you made and if the results were ok.
I hope you see this post and get back to me.
- Mar martinez pastor added an answer:Why are S. cerevisiae-transformants producing colonies, but the test PCR of the transformants is the same size as the WT band?
We have currently trying to add a GFP tag to a gene buy using homologous recombination with a PCR product. The marker we are using is G418 resistance and the transformations are done by a normal lithium acetate protocol.
We are seeing several colonies appear on our selection plates, however, when we go to test our colonies by PCR (using primers outside of the gene that is being tagged with GFP), we are seeing a band appear the same size as WT instead of substantially larger as it is supposed to be if GFP has been attached.
Every colony we have tested has turned out this way in several rounds of transformations and several different genes that we have attempted to tag.
Any help that anyone has would be greatly appreciated!
Try also to add the GFP tag in the other extreme (C-terminal or N-terminal). Some proteins are not functional with epitopes and can eventually release them. In this case, you would transform cells (you would be able to see colonies) but you would lose the tagFollowing
- Yuviraj Y A.Putten added an answer:Can I have your opinion whether the results of my gel electrophoresis are wrong or not?
I ran a gel electrophoresis at 1% agarose for genomic DNA of 2 endemic plants. I did a duplicate. The first two lanes on the left are the first plant. The 2 lanes on the right are the second plant. I ran the gel at 115 Volts for 40 minutes.
Ok sir , this is really helpful. I will do an RNAse treatment. Its for my final year project. ThanksFollowing
- Maryam Aminizadeh added an answer:What are the effects of carbohydrates in PCR?
I would like to know more about presence of sugars, or in fact all carbohydrates in PCR.
What are their effects? What should we do to remove them from the PCR reaction?
Thanks Dr Theodorakis.thanks for your full answer.
thanks dear Dr Ravi Kant Upadhyay and Dr Aglieri.Following
- Avin Koh added an answer:Will the agarose gel electrophoresis be affected by higher concentrations of EDTA in my TAE buffer?
If the concentration of EDTA (0.5 M) in 10x TAE buffer is increased, will it affect gel electrophoresis and consequently, the DNA bands when I use my 1x working solution? E.g. 0.7 M, 0.8 M, etc.
I see, thanks for highlighting that.Following
- Yahia Noureddine added an answer:Are there any other methods besides silver staining to visualize SSR PCR product in polyacrylamide gel electrophoresis?
Are there any other protocols?
Make a bath of EtBr and put directly your gels insides and than visualized your gels under UVFollowing
- Hayley Brodrick added an answer:Can I do an RNase A treatment post genomic DNA extraction?
I have some bacterial genomic DNA extracted using the QIAgen QIAextractor. When I ran it through a gel, it also had RNA in it.
This is new as we have just had our extractor upgraded, and something about the new reagents now allows RNA through. Can I treat the already extracted samples with RNase A rather than re-extracting and adding it to my lysis step?
They are going for whole genome sequencing. I don't mind redoing them, but it would be better if I didn't have to!
Any ideas would be great.
Thank you all!Following
- José A. Campos-Sandoval added an answer:Is there a good protocol for ion exchange chromatography and SDS-PAGE?
I would like to purify a plant extraction using the above methods.
If you want, you can write me to my email address and I´ll send you standard protocols for both methods.
- Alexandra Müller added an answer:What are the causes of a bad Western blot result?
I did the Ponceau on the last run, and it came out looking like these two images, attached.
What could cause this, besides bad gel?
You can get rid of DNA by sonicating the samples after adding SDS sample buffer and boiling. A sanitation bath is best-suited for this purpose. Sometimes it helps to reduce the protein concentration by adding more 1x loading buffer and then loading twice the amount of sample to each pocket. Also additional boiling/vortexing circles may help.Following
- Gulnaz Bashir added an answer:Why do the bands towards the electrodes move slowly in my electrophoresis unit?
I have recently purchased a new electrophoresis unit. On electrophoresis in 1 and 2% agarose, gel bands of same size on the electrode side move slowly giving the whole thing a slanting look. The unit had been properly leveled before. Can any body tell me the reason for that?
I am sending the photo of the apparatus and the gel picture as an attachment.Following
- Jürgen Denecke added an answer:What might be the reason for the bad sample migration in my western blot?
Recently I tried a western blot on various human muscle samples. Unfortunately, some of the samples migrated like the fourth sample you see in the picture. It seems like the proteins in the sample are too aggregated, but I really don't know what to do to "unpack" them and no one in my lab can give me any advice about it. I used RIPA lysis buffer to lyse the muscle sections, and broke them down by freezing and thawing them a few times.
Any advice would be immensely appreciated.
When you make protein extracts from different tissues, you should first normalise them by total protein concentration (before adding the sample buffer). Invariantly, when using manual grinding, or even freezing/thawing, the extraction efficiency is not the same, so you need to quantify total protein levels in your various samples. Afterwards, you bring all concentrations down to the lowest in your series by appropriate dilutions. Then you add equal volume of diluted samples and sample buffer, boil and run the SDS gel for later Western analysis. This means, when you run the SDS PAGE, you load equal voilumes and equal protein concentrations in each lane. If you just measure protein concentrations, and load different volumes to achive the same amount of protein in each lane, the samples will not run well. Always equal volumes and equal protein levels, it's more work, but it is necessary.Following
- Bhavin Uttekar added an answer:Do you think DNA/RNA used in experiments must be fresh?
Do you think after RNA extraction is better to synthesize cDNA immediately? Do you know cDNAs that were made after RNA extraction have a better quality than ones obtained from RNAs were hold in -80 oC for a long time? As a whole do you think
for DNA/RNA, frequent thawing cause to quality decreasing? any idea about this?
You know I did DNA extraction, it was ok but after about 3 month I had to do gel electrophoresis again but it was not good at all. Do you have any reason for it?
Let me discuss you the problem i faced when RNA was thrown out of the -80 by someone unknowingly. The amount of RNA was reduced after 6hours by 400 units less than as it was initially. So immidiately prepare cDNA. Try to reduce freeze thaw...Following
- Dacquin Kasumba added an answer:How can I separate agarose gel electrophoresis close band sizes?I have a clone cut by an enzyme and the result of that cut is two bands, which are 12000 bp and 8000 bp. On 1% agarose I was not able to isolate DNA from gel because they appear at the same site on gel. Does anyone have an idea how I can isolate the 8000 bp band from that gel?
as suggested by others try to use lower percentage for your gel, run at lower voltage and load less DNA on the gel. I have attached a guide that could be useful...
- Tikam Chand Dakal added an answer:Running nice histone protein gels?As part of my experiment I was trying to run whole cell extracted protein lysates on glycine SDS-PAGE gels, 12% and 15% and probing for total histones, phosphorylated histones, and acetylated histones. I was getting nice acetylated histone bands but when I started probing for phosphorylated or both p and ac histone I've got a huge background, very fuzzy smear bands and no clear single band. I have read that there are specific methods for extracting histones with acid and running them on urea gel but don't have a good protocol or any experience with that. I also tried running the samples on gradient tricine gel but this didn't help too. I would be very happy to hear any suggestion on whether I should change my extraction method and/or running method etc.
Dear Dr. Panigrahi,
The information you provided is really useful.
Thank you very muchFollowing
- Ajit Kulkarni added an answer:What is the best replacement for ethidium bromide gels?
I want to use safe and non toxic gels for electrophoresis. I found couple alternatives but shelf life is too short and they dont have many pre-cast gel options. Any suggestions will be greatly appreciated.
We also use the SYBR safe (invitrogen or Life technologies).Following
- Fan Zhang asked a question:Among gel imaging systems, what advantages does the Bio Rad XRS+ have over the UVP biospectrum 510 system?
The XRS from Bio Rad is a little expensive for us but it has been widely used by other labs. What is the advantage of XRS?Following
- Dr. Godfred A. Menezes added an answer:Can you share the crucial key points in PFGE optimising?
Crucial key points in PFGE optimising.
Thank you so much Lorraine. I will get back to you.Following
- Jhoti Somanah added an answer:Does anybody have any experience with using comet assays?
Have some basic queries regarding initial setup:
1) Do I need to use the microscope in fluorescence mode?
2) If I put my stained slide on the stage, will the image on the screen remain black, or show me fluorescent cell nucleus?
Any tips will be greatly appreciated!
Matheus and Tanya, your advice is highly appreciated. Thanks very much.Following
- Anjana devi Tangutur added an answer:Have you ever seen the pipette tips for cutting from the gel?
My advisor told me he had some sheet with ad for pipette tips used to cut bands from gel. However he didn't have it anymore so he couldn't tell me the official name nor brand.
Have you ever seen anything like that?
Yes, we do use Axygen tips for cutting bands or spots from PAGE for proteomic analysis.Following
- Marcin Schmidt added an answer:What is the minimum DNA quantity (ng) required to visualize DNA on Agarose gel?
I have very low amount of DNA 10 ng/ul. So if I would like to check on gel, how much ul I have to take?
How much is visible depends also on transiluminator and fluorescent dye you are going to use. UV is for EtBr, SYBR dyes, however other dyes (there is lots of them) may require blue light - check specifications.Following
- Alexander Streng added an answer:How can I explain a non-specific band on a western blot that appears with several different probings of different species?
I have been reprobing the same two blots most of the week and keep noticing a band in the 50-75kD range that doesn't correspond to anything I'm probing for. What's even more odd is that it appears when I probe with mouse monoclonal or rabbit polyclonal and bovine-anti-mouse or goat-anti-rabbit, respectively. Can anyone explain how that's possible?
In my experience, aspecific binding of that sort is usually either Albumin or IgG. It depends a bit on what sample you are loading, so a bit more information on your sample preparation may be helpful. Also, can you be a bit more specific on which bands you're seeing on which mass ranges (for example add a picture)?
You may want to deplete your sample first using immunoprecipitation or a depletion kit of some kind to get rid of abundant proteins. Diluting your sample a bit more may also do the trick, but that all depends on how strong or faint the bands are.
Also, in my opinion reprobing blots leads to very dubious results as you can never be certain you have stripped your previous antibody completely. So, it may also be possible that you're seeing carryover from one of your first blots. Does the signal keep constant or does it diminish with each probing step?Following
- Vakamullu Sreedhar added an answer:Dose anybody know how many cell i need seed in t25 flask for detecting dna laddering after 72h treatment?
i work on sw742 colorectal cell line
1X106 cells are seeded in T-25 flask for detecting dna ladderingFollowing
- Anant Dave added an answer:For identifying an unknown protein under specific induction condition, should we use a 2D page or mass spec?
I would like to know which one is more efficient. I know 2D page is error prone and time consuming.
Hi Youli, I agree with Peter and recommend doing both. The MS can identify the sequence, as most people have pointed out but doing a gel will also let you determine specific linkages such as disulfide bonding which otherwise may be missed by MS. The MS exclusively requires the disulfide bonds to be reduced otherwise the estimation of molecular weights could be difficult. Indeed the in-gel trypsin digestion followed by MS/MS seems to be the best method but its efficiency depends on how well you are able to extract the peptides from the gel matrix. Good luck!Following
- Anna Git added an answer:Can anyone help me with gel electrophoresis of total RNA?I want to check the quality of RNA on non-denaturing gel. I found this method: incubate 2 ug RNA with two volumes of denaturing buffer (50 ul formamide, 20 ul formaldehyde, 10 ul 10 X MOPS, and 2 ul ethidium bromide) denature at 70C for 3 minutes and immediately place on ice. Then run on 1.2% TBE agarose gel at 90V.
Did anyone try this method or have a good experience with alternative method?
They are absolutely perfect in every possible way, well done :-)
28S/18S ratio is about 2, tRNA/5S band is clearly visible, no background smear.Following
- Azura Amid added an answer:Are there any methods to amplify unknown proteins using PCR?
We are using beetles within our experiment and having a difficult time extracting enough protein to obtain a good gel electrophoresis result. I am wondering if there is a simple process using PCR to obtain more of these unknown proteins? Or any other advice would be great! Thank you in advance.
You can not isolate protein by PCR because PCR amplify DNA and not protein. Normally we already know a bit of the DNA sequence of the gene of interest. If you would like to use the same concept you must know a bit of the amino acid sequence but yours is unknown protein.Following
- Yuan-Yeu Yau added an answer:What is the definition of Monomorphic, polymorphic or unique bands in gel image?
How we can define in simple words the differences between Monomorphic, polymorphic or unique bands on gel image can from gel electrophoresis
If you have 30 samples on a gel (for example, PCR a gene from 30 plant species), 29 samples have 0.5-kb band in size, only one sample has 1-kb band, I would consider this 1-kb band a 'unique' band. It is unique to other samples and we don't know what happen to this gene locus.Following
- N.Subash Chandra Bose Raju added an answer:Cheap and most suitable DNA purification method or kit?Can anyone suggest a cheap and the most suitable DNA purification method or kit?
Dear Suni Ge,
Please use DNA investigator Kit -Qiagen which is promising Kit for dry specimens.This might be delayed reply but useful for many dry specimens.Following
- Sanjay Kumar Singh added an answer:How can I determine the Shannon Wiener Index of a DGGE profile if some distinct bands on the gels represent same bacterial species?
In DGGE analysis Shannon index works on binary data. In my case, some of the distinct bands turn out representing the same identical bacterial species after sequencing.
In question I could not find any thing about 16S rDNA gene. Additionally there are always certain assumptions with any technique. When sample numbers are very high it could be used for removing similar profile samples. Technique is good but not perfect for community analysis.Following
About Gel Electrophoresis
Gel electrophoresis is a method for separation and analysis of macromolecules (DNA, RNA and proteins) and their fragments, based on their size and charge.