DNA Purification - Science topic

Hello,

I'm trying to clone some short genes *up* to 350 bp into an expression vector the clasiccal way. For this, I do PCR with Q5 and get (mostly) nice band. I cut it from the gel and purify using Macherey-Nagel kit.

I digest this DNA with BamHI and either HindIII or PstI. The first time I aimed at 2 ug, the second time I simply digested all DNA. I run the digested DNA on gel again and purify with the kit again.

After this I get low concentration (5-40 ng/ul) and often poor purity (A260/A230) way below 1.8 (I attached a picture of one such sample, where I had actually peak at 230 nm; that's one extreme). The concentration of plasmid is repeatadly in the range 6-10 ng/ul (it's 5.5 kbp).

Any idea, what could be the problem? In other projects, I usually have sufficient purity and reasonable yield after gel-purification.

Thank you

What would be the best way to preserve exhumed human bones (in archaeological or forensic contexts) for downstream decontamination and genetic extraction + analysis?

For example, a specific analytical grade buffer or other chemical solution to submerge the specimen, the most ideal temperature (RT, -20C, -80C, etc.).

We have performed CHIP assay using magnetic bead CHIP assay kit (Cat: 9003, CST) and got desired band in agarose gel electrophoresis after the chromatin digestion. But when we went for RT-PCR did not get proper data. Can somebody suggest me how to troubleshoot this problem? After immunoprecipitation and DNA purification we got the DNA concentration 18 ng/ul. We did not get any amplification even in 2% input sample.

I have issues with the 1D ligation protocol of the MinION sequencer when handling high molecular weight DNA (>50kb). There are several cleaning steps with AmpureXP beads and the DNA is so viscous that I can't properly recover the DNA (low recovery and substantial loss of HMW over LMW DNA). Sometimes the DNA clumps when mixed with the beads and most of the times DNA get stuck on the beads (the magnetic field is not strong enough to retain the beads when pipetting the solution). Does anyone have any tip to share please? Thanks in advance.

Hello,

I am currently facing challenges with DNA extraction and subsequent PCR amplification for barcoding insect beetles. I am using Chelex 100 resin for DNA extraction with the following protocol:

I am looking for insights or suggestions on how to improve DNA purity and enhance the efficiency of my PCR reactions. Additionally, any advice on barcoding from the article "Barcode 100K Specimens: In a Single Nanopore Run" would be highly valuable. Could the low purity ratios be affecting my PCR efficiency, and how can I optimize my protocol for better results?

Add aditional infromation about primers.

Best regards, Benediktas

I have used the QIAGEN DNeasy kit to obtain fecal prey DNA from an herbivore and a few of the samples have higher polyphenol levels, which have given a yellowish tint to the final eluted DNA solution.

Should I go ahead with Geneclean (suitable for 200–20 kb) DNA purification from solutions or the traditional ethanol and sodium acetate precipitation to remove the PCR inhibitors in the form of polyphenols and other plant secondary metbolites?

Dear colleagues

I have a GeneRead QIACube station which is specified for no longer supported Qiagen NGS GeneReader workflow (emulsion technology). It looks pretty much the same as casual QIACube, but has other worktable and screen. I just can't put a standard reagent tray into the device.

Is it possible to convert GeneRead QIACube (NGS sample preparation) into a QIACube for NA isolation?

I'm currently working on gel purification in order to get DNA fragment for T4 ligation. But I have been able to take only 8.5ng/μl as a highest concentration of DNA fragment. And could not get ligated plasmid with that fragment and other fragment(other fragment have high concentration, good waveform on nano drop so I don't think the fragment is a cause of failure on T4 ligation.).

Feel free to ask me question for my experiment.

Thank you.

Support information : I will show my protocol briefly as a example for DNA fragment which I could get highest concentration. I cut 4μg of plasmid with enzyme and did electrophoresis them and separated DNA fragment. The band was clear and cut band on edge. I did gel purification with them.

Expected DNA fragment length is about 8600bp. I tried elusion method with warm elusion buffer(70℃) and put column on 70℃ for 5 min just before centrifuge. I'm using E.coli DH5α for transformation of ligated plasmid.

P.S.

I changed only competent cell strain for my protocol and I could take colonies. Result of colony PCR suggests that I may get optical plasmid. Of course, I will do sequencing later for my plasmid. (2024/8/8)

Using INTRON's DNA-spin Plasmid DNA purification kit to extract plasmids from specific E.Coli strains, my team and I have encountered numerous plasmid dimerizations that lead to over-weighted plasmid DNAs which are hard to identify whether they are the wanted plasmids or other common impurities. We have followed the protocol given by the producer but have not achieved notable success under our lab conditions and we are needing help right now.

I have extracted historic DNA from animal tissue using the Qiagen DNeasy kit and eluted the DNA in the provided elution buffer, which contains EDTA. I will perform whole genome sequencing on an Illumina platform. Because of the low DNA yield, I will utilize the TrueSeq Nano or Microplex prep kit. These library prep kits are sensitive to EDTA. I must therefore cleanup the extracts and use an EDTA-free elution buffer. What approach should I use for the cleanup? 

I will avoid AMPure cleanup due to the small peak sizes (<150 bp) in my DNA templates. Grateful for all suggestions!  

I currently need to purify DNA after plasmid linearization. I am using the TaKaRa DNA purification kit, but the concentration of the diluent obtained from each DNA purification is very low. Do you have any other recommended kits?

I typically extract DNA from whole blood using a Qiagen kit link here: https://www.qiagen.com/ja-us/products/discovery-and-translational-research/dna-rna-purification/dna-purification/genomic-dna/qiaamp-dna-blood-kits/ (RBC lysis, cell lysis, then within 2 years, protein precipitation, DNA precipitation after adding isopropanol, wash with ethanol, rehydrate DNA). I want to extract DNA from PBMCs which have been frozen in aliquots of 5 million cells in media comprised of 90% FBS, 10% DMSO. I've tried this before by thawing the PBMCs and proceeding onto the cell lysis step in my normal DNA extraction protocol but have had no success. Can anyone offer tips on how to extract DNA from frozen PBMCs? Thank you in advance!

We used the DNA Clean & Concentrator-100 (D4030) from Zymo Research to clean and concentrate our 100 µg DNA. Unfortunately, our yield was only 8%.

We initially believed that, due to our DNA being over 10 kb, it was not eluted properly from the column. However, upon checking the DNA in the flow-through and wash samples at each step, we discovered that most of our DNA did not bind to the column. Specifically, 90% of our DNA existed in the flow-through. When we applied the flow-through containing this DNA to another company's DNA binding column, most of the DNA bound well to the column. Consequently, we suspect there is a problem with the DNA binding column of the Zymo kit.

Has anyone had a similar experience? If so, how did you resolve the problem?

I am trying to clone a large (11kb) and toxic gene in an expression vector. Therefore, I am using the EPI400 CopyCutter strain that is maintaining the plasmid at a low copy number to allow bacteria growth. Attached is the protocol for this strain.

I have to grow the bacteria on plate at 28 C for 2 to 3 days to be able to get colonies.

I did a colony PCR and got 20 positive colonies.

When I put them in liquid culture (5mL to 10mL), some colonies never grown, even at 28 C. Some of them did grow (2 days incubation 28C, 250rpm in aeration culture tube, OD600 above 2.0), but then I only get a really low yield after induction of the high copy production in 100mL culture in flasks.

I tried this several times but still got a really low yield (10ng/uL, maximum 15).

I used this strain for other toxic genes and was able to get more than 40ng/uL (which is around what I need to be able to sequence my plasmid and verify the correct insertion of my gene).

I am using this kit for plasmid extraction Wizard® Plus SV Minipreps DNA Purification Systems

Any suggestions on how I could improve my plasmid yield?

Any suggestions to be able to grow the bacteria in liquid culture after getting them selected on the plate?

Thank you.

I am getting zero DNA yield after using qiagen purification columns. I finally traced the problem to NEBuffer 3.1, but pH doesn't seem to the cause.

Essentially, I observe:

3 ug of DNA in 50 uL of water ->

qiagen purification column ->

1.5-2.3 ug of DNA

In comparison:

3 ug of DNA, 5 uL of 10X NEBuffer 3.1, bring to 50 uL with water ->

qiagen purification column ->

zero DNA

I thought it was a pH problem -- high pH can cause low efficiency. But I don't think pH is the problem. Because pH strips and qiagen's pH indicator say my pH is okay (pH<7). And I added 20 uL of 3 M sodium acetate (pH 5) and it doesn't fix the low yield at all. I observe:

3 ug of DNA, 5 uL of 10X NEBuffer 3.1, bring to 50 uL with water ->

Add 20 uL of 3 M sodium acetate (pH5) ->

qiagen purification column ->

zero DNA

Why does adding NEBuffer 3.1 cause low yield if not pH problems?

I am looking for a protocol which I can get the purified DNA from FTA cards easily and inexpensively.

Then I want to use TE buffer instead of using buffer AT, ATL.

The thing I want to know is that if buffer TE can work instead of buffer AT, ATL or not.

Good morning everyone!

I am a Biotechnology student and I am doing a project to verify the biocontrol capacity of actinobacteria, for this I must carry out the molecular identification of the strains.

Is it advisable to use any of these DNA extraction and purification kits?

- Wizard® HMW DNA Extraction Kit from PROMEGA

- Invitrogen™ PureLink™ Microbiome DNA Purification Kit

Is another type of kit more recommendable?

I am currently working in the process of automating our research lab to be able to process a higher number of samples. Our work consists in detecting and quantifying pathogens in mammalian samples (e.g., blood, faeces, tissue, swabs). As such, I am interested in an automated extraction system which produces good DNA/RNA yields to be sent for NGS.

Until now, we have been using Qiagen kits for our manual extractions, so I thought that a machine from the same brand would do for us. However, I've been told that the QIAcube Connect does not really take that much work out of your hands, and that the sample volume obtained at the end of the process with the QIAcube HT is way lower than the one with the manual kit. I have also checked other machines, such as Thermo Scientific's Kingfisher Flex and its kits, but do not know how well they do in comparison with Qiagen's kits.

Based on your experience, which automated extraction system would you recommend? And which brand of kits have you used with it? The system and kits do not need to be from the brands mentioned here (as long as the produce good results).

Thank you very much in advance.

Amplified PCR product is clear as 1 band in gel. but Ethanol precipitation or magnetic beads purification of same PCR product resulted in extra unknown band. why?

I'm a grad student working on metagenomics and I ran into some issues with the samples. I collected urine samples and extracted DNA using DNeasy Blood & Tissue kit. I used NanoDrop to measure DNA concentration and ran into different issues. The problem is that the A260/280 level is too low (not to mention the contamination) and I don't know how to increase the purity level. I don't think I can collect the samples again, or at least it will take a while before I can do that again. Is there a way or a kit I can use to increase the purity level of my DNA extracts without significantly lowering DNA concentration?

lac promoter is widely used in plasmids. It seems strange plasmid vector for DNA purification contains the CAP binding site and lac promoter. Both pLKO.1 puro(https://www.addgene.org/browse/sequence/333600/) and lentiCRISPR v2(https://www.addgene.org/browse/sequence/244694/) but not pLL3.7(https://www.addgene.org/browse/sequence/237575/) contain the lac promoter.

so is it safe to delete the cap binding site and lac promoter sequence in lentivirus vector, or even in some other mammalian expression plasmid like pcdna3(https://www.addgene.org/browse/sequence_vdb/2092/)?

Hi,

I wonder if anyone knows how to use sample release reagent from Sansure Biotech ?

Hi,

Recently, our workflow stated below has stopped working.

-Sample Prep-

Swabs taken from mouldy plasterboard.

1. DNA extracted using Genomic DNA purification Kit and eluted into gDNA elution buffer. (2ng/uL)

-Whole Genome Amplification-

2. 1uL samples amplified using UltraFast REPLi-G Mini Kit. (1.5ug/uL)

-T7 endonuclease treatment-

3.7uL amplified sample + 3uL NEB Buffer 2 + 1.5uL Endonuclease T7+ 20.5uL diH2O 4. 15 mins @ 37 degrees 5. 18uL TE Buffer 6. 35uL AMPure XP 7. Magnetic bead clean-up (2x 80% EtOH washes) 8. Elute into 50uL diH2O <----- LOSE ALL OF OUR DNA (<1ng/uL remaining!)

-Nanopore Sequencing-

We have replaced all chemicals/ reagents from buffers to AMPureXP beads and nothing seems to work.

We have increased ethanol washes from 70% to 80% to ensure the DNA isn't being eluted off too early.

We have tried replacing the AMPureXP beads and alternative SPRI beads but the results are the same (no DNA!).

AMPureXP beads work for the clean up step during ONT RBK004 sample prep.

We have tried messing around with the pH of TE Buffer, from pH4 to pH8, to see if the DNA binding to the beads is influenced but this doesn't seem to help.

Any ideas? Has anyone overcome any similar issues with AMPureXP beads?

After adding resuspension buffer and lysis buffer to the cell pellet, the next step is the addition of neutralisation buffer which requires immediate mixing. The expected outcome is the formation of large, fluffy and white precipitate. Slight delay in mixing results in the formation of small floating precipitate which remains suspended despite doubling of centrifugation duration, resulting in the decrease of A260:A280 eventually.

What are the remedies to solve this issue and improve the DNA purity? The DNA is plasmid DNA. Thank you.

Hi!

I am about to use 95% formamide, 10mM EDTA buffer to elute biotinalyted DNA from streptavidin bead. So should I just simply add 1ml of 1M EDTA, 4ml of water and 95 ml of formamide to present 100ml of the elution buffer?

Thanks!

Hi everyone

I'm processing some leaf samples collected in different rivers for shotgun metagenomic sequencing (Illumina NovaSeq 6000).

The company that will sequence my DNA samples (Novogene in UK) requires a 260/280 ratio =1.8-2.0 (no degradation or RNA contamination).

(Novogene webpage:

https://en.novogene.com/services/research-services/metagenome-sequencing/shotgun-metagenomic-sequencing/)

But I've sent samples in the past (to the same company but for amplicon sequencing) with 260/280 = 1.7-1.8 and they passed the quality control and went full analysis.

Regarding the 260/230 ratio, they do not refer any requirement but my 260/230 ratios are even lower (the lowest is 0.6).

I'm using the DNeasy PowerSoil kit (Quiagen).

So my question is, how low can these ratios be for shotgun metagenomic sequencing?

What's the limit?

I know that I will probably have to clean some samples but I just want to have an idea to help me select the ones I definitely have to clean.

Thanks a lot!!!

I've been having some trouble with RNA contamination after plasmid extraction with the Qiagen mini-prep kit.

The 260/280 ratio looks fine after extraction, and I have been having this problem only with 2 out of my 6 plasmid samples with the same bacterial strain.

What could be the reason for RNA contamination aside from excessive starting material, expired RNase, and RNA contamination of buffers?

Also, I would appreciate any kind of tricks that you personally use to avoid RNA contamination.

I purified about 5 uL of amplicon from about 1 month ago, but after running the quantititation gel, I don't have enough remaining to ideally perform my downstream applications (Sanger sequencing). I still have remaining amplicon which I have now purified once again using EXOSAP-IT. Should I run quantititation gels on this product as well, to confirm the concentrations? Ideally, I would like to simply rely on the results of my first quantititation gel from this amplicon.

The amplicon has been sitting in a +4 fridge for about a month, undisturbed. Please let me know if there's any additional information or details I can provide to help inform an answer - thank you in advance!

I am currently working doing a lot of N/N 2D gel electrophoresis for investigating replication structures in yeast. We might invest in a bead beater, which can handle a specific volume, but I am afraid that bead beating instead of vortexing will shred the DNA structures. Vortexing is time consuming, hence considering.

My experiment requires to extract Tissue DNA from zebrafish intestine , this is my first time to learn Tissue DNA extraction. Recently I have learned about extracting DNA using magnetic beads, but i need more recommendations and and what is your experience about magnetic DNA extraction technique and how many samples can be extracted from single kit. Beside this , please can you suggest me any other methods that follow more simple steps.

Hi RG members,

I found NTB buffer suitable to extract chromatin in buffer containing 1% SDS used for ChIP-seq experiments. I am wondering if anyone knows what is the composition of NTB buffer.

Best,

BS

Hi everyone. I know it sounds weird but I was doing a midi (qiagen) prep yesterday and just after the last centrifugation, all isopropanol-eluted DNA just dissapeared. I saw the cloudy phase forming before the centrifugation step and when it finished I literally saw no pellet at all. My best guess Is that pellet was formed but is really llittle and I just didn't see it.Any opinions? I kept the tube at -20°c for trying again on Monday. Thanks again if anyone can share their theory about this !

Hi,

I work with voltage-gated sodium channels, which are a pain to clone into high-copy number plasmids. Recently I have acquired the pBI-CMV1 vector, which is a low-copy number vector, and it has really helped with the cloning. However, when it comes to DNA purification, I have had lots of issues. Sometimes the DNA yields are extremely poor (I mean barely detectable by Nanodrop), despite longer growth periods and increased culture volumes/lysis buffers (Qiagen) compared to high-copy number plasmids. What is most worrying is that I often get a high level of "smeariness" when I run miniprep/midiprep pBI-CMV1 DNA on an agarose gel. It's not one distinct form of contamination; it's more of a background smear/smudge that stretches across all DNA sizes on the gel (and this can be seen even without adding restriction enzymes). It makes Nanodrop yields deceptively high and ruins the DNA for downstream applications like transfection. I'm not quite sure what's causing it, as I've never seen it with my other cloning/expression plasmids. I do not use endA+ bacterial strains for cloning. Has anyone worked with pBI-CMV1 before? Or has anybody encountered a similar problem with a different plasmid and been able to resolve/minimise it?

Thanks!

Is it ok to use water for injection (WFI) instead of ddh2o for molecular biology work, such as pcr or dna purification?

Every time I try to isolate DNA, it ends with a smear like band on gel, or the pellet obtained is very little. Currently I am using a protocol which requires 2 days to isolate DNA.  If possible please suggest a protocol that enables isolation within the same day.

I am preparing 4C library but i think there is a problem of excess amount of DTT of ligase buffer that interferes with evaluation of DNA concentration by nanodrop.

Because for doing PCR of 4C library we need to know the exact con. of DNA so,i am looking for a extra or updated technique to remove or decrease the DTT content before, during or after 4C library purification.

Hi everyone,

I need to clone in pGEM-T vector the result of amplifying a metagenomic library by PCR with a high fidelity polymerase but I only want to clone the fragments of an specific size (1-4kb). The rest is not interesting. I was thinking about:

-Purifiying this PCR product

-Do a A-tailing

-Run an agarose gel to cut the band of the desired size

-Perform pGEM-T clonning

I am not sure if A-tailing will survive the whole gel process. Any idea or help with this?

Thank you so much beforehand.

I need to extract large amounts of plasmid DNA for transfection into mammalian cell lines with as little as possible enterotoxins and I usually use the Qiagen Endofree plasmid maxi kit for the extraction. When doing miniprep with the Qiagen plasmid miniprep kit extractions to select colonies for the maxiprep I get excellent yields of over 300ng/ul with a 60ul elution. Since returning to the lab in 2021 I have tried a PureLink™ HiPure Plasmid DNA Purification Kit and the Qiagen Endofree plasmid maxi kit and got less plasmid out of these kits than from the miniprep kit.

Do you have a possible reason for the low yields and suggestions for me to improve the yields

I have been trying to extract DNA from above mentioned sources using different CTAB/SDS Buffer Compositions and purifying agents but I am not able to extract DNA from roots of :

- Dactylorhiza incarnata

- Nannoglottis hookeri

And Barks like:

- Cinnamomum verum

- Santalum album

And Galls like:

- Tamarix indica

- Pistacia chinensis

I want to use DNA EXT Kits except DNEasy that are a little cheap and please recommend any.

Please share your valuable experience to solve my problem.

Thanks in anticipation.

Kind Regards

My PCR product has smear when I run it in the gel. I would like to use purification kit and run it again. Is this way useful for removing the smear?

Hi everyone,

I perform a PCR + Restriction Digestion (single cut, same at both sides) over a metagenomic library. After that, I am trying to purifiy DNA fragments between 1-4kbp long from the smear I obtain after running the PCR in an agarose gel. Agarose gel is 0.8% and I run it at 100V 40 minutes. I carefully cut the band between 1-4kb and the yield or DNA recovery is quite good. The problem is that, after cloning it and taking a look at the size of the inserts, 90% of them are much smaller than 1kb.

What would be the best DNA Electrophoresis conditions (% agarose, Voltage, time ...) in order to maximize the separation between 1-4kb fragments from the smaller ones?

I believe that even a small quantity of small fragments after purification will be over represented after cloning protocol so, every tip in order to get rid of them as much as possible will be very welcome.

For tIn the ligation step I tried different ratios vector/insert (3:1 ,1:1 and 1:3) and temperatures (RT, 16 and 4ºC). The size distribution is basically the same.

Thanks a lo beforehand

Jorge

Does anyone know the composition of the Buffer MP that comes with the Qiagen M13 purification kit? I gather by the MSDS that citric acid is a major component.

I work as a lab tech prepping university classes; I don't get a lot of opportunity to get some microbiology experience, and I thought I would take advantage of the extra down time we have since the semester was ended early.

I took on a little project of seeing what kind of bacteria are growing on my pet chinchilla. I do not know what the bacteria is.

I have 5 samples; 4 are gram-positive coccus bacteria and one is a gram-negative rod bacteria after doing a gram stain. The 4 coccus bacteria are circular colonies, ranging from white-ivory-orange. The rod bacteria appears to be a rhizoid colony that is semi-transparent (these were all grown on YPD agar plates).

I just followed the Qiaprep spin mini prep protocol where I used 2mL of bacteria to extract DNA. Because I am unsure what exactly the bacteria is, I decided to do the step where you wash the spin column with 500uL of Buffer PB.

What should my agarose gel and DNA sample that I load look like? I want to see if the extraction was successful.

This is my second attempt at DNA extraction for this project; the first time I ran a 2% agarose gel with sybrsafe at 180V for 40 minutes, using 6uL of a 1KB ladder and 1uL 6X LD/5uL DNA sample (extracted from 1mL of culture) and only got a single, extremely faint band. I thought maybe the sample was too little, so I tried it again at 3uL 6x LD/15uL DNA sample but got the same result.

I've been able to understand and kind of make my own protocol up till now; I'm proud of myself for doing a successful gram staining, considering it was my first time ever trying it (btw, neutral red seems to be an alternative counterstain if you have no safranin or fuchsine available). I just don't have a lot of experience working out these kinds of problems (hence why I'm trying this). Is there something I'm doing wrong here and how should I proceed to get a successful DNA extraction?

I just bought a plasmid from addgene, the name is DMP1-GFPtopaz.

However, I could not extract the vector DNA by using Qiagen DNA purification kits from the bacteria.

the bacteria growth was very well at 30 ug/ml carbenicillin.

I know the vector is very big.

I wish someone has handled this plasmid and can give me some suggestions to fix this problem.

Thanks!

Hello !

I would like to extract gDNA for soil to do NGS. We have the QIAGEN Powersoil kit. I did it twice now. First, according the kit instruction : concentration = 10 ng/uL, 260/280 = 1.4, 260/230 = 0.7. And the second time, I changed the lysis step by vortexing for a longer period and I heated the elution buffer at 50C as ssen on other discussion but my results are still not that good : concentration = 10 ng/uL, 260/280 = 1.8, 260/230 = 0.8.

Do you maybe have an idea on how I can improve my ration to send the sample for NGS?

Thank you !!

We are extracting DNA from frozen (a few years old) human blood samples using a Qiagen mini and blood mini kit. We usually get okay to good yields afterwards on the Qubit (anywhere between 10 ng/ul-over 100 ng/ul, its been fairly inconsistent). We then usually try to run PCR on our DNA yields that are higher. The primers we are using are all between 200-300 bp and we have a general but slightly altered PCR cycle for them. Our gel results are inconsistent, sometimes look great but sometimes nothing will show up or bands will be really faint. Our purification using a Qiagen QIAquick PCR purification kit has never had a very good yield. The most we have gotten from it is around 10 ng/ul according to the Qubit, but usually we get under 5. We need at least 20 for sequencing. We have tried a ton of things but are really at a loss right now for what could be causing these problems.

I spiked in 4-8 log CFU/ml of Lactococcus lactis into milk, and use DNeasy Powered Food Kit to extract DNA from it. Nanodrop 2000 was used to determine the values. The yield and quality are weird: the concentration of DNA was only 0.5-1.6 ng/ul, and 260/230 is 0.11 to 0.58. Both I and the lab assistants got similar results. We followed exactly the manufacturer's protocol.

I am simply trying to obtain high yields of DNA from fish scales (they are tiny, like 1mm each).

The idea would be to obtain a good amount of DNA with fragments of 4KB at least for amplification of mitochondrial genome.

As a reference, the Wizard SV 96 Genomic DNA purification system (Promega) results in very low concentrations of DNA (average per sample is inferior to 10ng/µL in a 75µL elution volume and seems highly degraded on agarose gel).

I am looking for a scalable approach (I still have hundreds of samples to extract) but I can afford to buy relatively expensive extraction kits or others if necessary.

So, what would be the best method for you?

Many thanks for your participation to the discussion, and I hope it can be useful to others too.

Have a good day,

Noé Barthelemy

I am using the 30-uL QIA gel purification kit, and each time I test the product on a nanodrop after, the 260/230 is in the 0.01-0.30 range. Subsequent reactions, like ligations have issues with extremely low efficiency. I typically elute with 30uL H2O. Asking around has lead me to believe that this means there is too much salt in my samples.

A Zymo Clean&Concentrate protocol afterwards typically works, but there's inevitable some sample lost when adding in extra steps like this.

I've tried all the usual recommendations (extra binding buffer wash, let wash buffer rest a few minutes at RT, extra ethanol wash, elute with warmed H2O), but nothing has worked so far.

Can anyone help me out with this?

Hi!

I use pyridine borane to react with DNA. I observed that DNA recovery on the purification stage after pyridine borane reaction is very low, at 10%. Therefore, I would like to kindly ask you about the following:

Does pyridine borane fragment genomic DNA?

Does it affect the efficiency of DNA binding to the column bed during purification?

Does anyone recommend a suitable purification method and tested binding buffers or other suggestions to improve DNA recovery?

Additionally, I have a question, the pyridine borane solution with DNA is shaken for 16h 850rpm, but I wonder if I can mix with vortex before incubation for better distribution of the substance in the solution?

I will be grateful for all suggestions.

I have some plasmids with sacB. Ecoli carrying these plasmids seems slow even on standard LB without sucrose.

I sequenced the plasmids and everything seems normal.

Normally I have nice colonies by ~14-18 hrs, but these plasmids require more like 24-28 hrs for nice colonies.

It seems even more dramatic for liquid cultures: I have to incubate them for ~24 hrs.

Is that normal?

Hello, I need to extract DNA and RNA from fish mature spermatozoa and unfertilised oocytes. I am also looking for a protocol to isolate mature spermatozoa from fish semen/testis and any advice on how to proceed with oocyte DNA/RNA extraction.

Normally, ecoli miniprep cultures are grown for around 12-18 hours at 37C.

However, due to my schedule, I won't be able to harvest the cultures for >24 hours. I think >24 hours is too long to grow ecoli cultures at 37C. I cannot freeze the cells and miniprep them later -- I will not be in lab at all for >24 hrs.

I want to grow them shaking at lower temperatures to slow their growth so I can harvest them later. However, I do not know what growth duration is appropriate at these temperatures.

How long should ecoli be grown at 30C and 25C to get something similar to 12-18 hrs at 37C?

Standard practice for plasmid minipreps is to grow ~5 mL ecoli cultures overnight (~16 hrs). This works well and I always get good plasmids.

However, a few times I wanted just *a little bit* of plasmid. So I only incubated for a few hours:

2 mL cultures for ~7-8 hrs at 37C.

The culture is a bit cloudy by this point and gives an okay yield of plasmid DNA.

However, this plasmid DNA is always seems very poor quality.

If I submit it for sanger sequencing, the results are a little ugly. As if I have a couple SNPs. A tiny bit of contamination. The plasmid is there, but it is dirty.

If I try to electroporate it into bacteria, it always "arcs." Like there is very high salt contamination.

I must re-transform it into ecoli and do a proper, 16 hour overnight culture to regain good plasmid. Then the sequencing is clean and perfect, and it electroporates with no arcing.

Why is this? Why do dilute, low-volume cultures always give such bad plasmid DNA? I would have thought "young" culture would be cleaner.

It is very frustrating because short miniprep cultures seemed so nice and convenient. But the prep is so dirty I think I am wasting my time.

As a technician I sometime want to compare different lab products. There are a lot of website and I am wondering which websites you use?

I want to maximize the yield of DNA extraction from insects. I am already using different DNA isolation kits and I know that these are not specifically designed for insect samples. Does anyone have any tips to maximize the yield?

Hi,

I am working towards standardizing and performing ChIP-Seq experiments on Arabidopsis thaliana seedlings and roots. The mutant line I am using is tagged to YFP. I use about 1g of plant material. Do a formaldehyde fixation (10 minutes under vacuum and 10 minutes without vacuum). I shear the DNA using a Bioruptor Plus (from Diagenode) – 30 seconds shearing – 60 seconds stop and 30 cycles. I get fragment size between 300bp to 1500bp. I do an IP with GFP antibody from abcam and Protein G dyna beads from Invitrogen. I use 10-20 ug of DNA for IP. After IP I do DNA purification using Qiagen Min-elute DNA purification kit. I end up with very low concentration (2-10ng/ul in a 10 ul elution with elution buffer) and low A260/230 values (between 0.5-1.5).

I had two concerns regarding this

How can I increase the shearing efficiency (to get sheared DNA between 200bp-500bp)?

How can I increase the concentration of the DNA after IP?

Any suggestions will be appreciated. Thank you

Hi,

I recently did an DNA extraction using the Qiagen DNeasy Blood and Tissue kit (protocol for insects). 260/280 values were okay, but 260/230 seemed a bit low (1.7). I still did an amplification and purified it with Promega Wizzard Gel and PCR purification kit. It seemed to have worked based on a bit of DNA I was able to run on a gel (I get the expected band). HOWEVER, when trying to load samples (with loading buffer) on a gel a lot of the sample did not sink into the pocket. Also 260/230 values are really low (1.20 - 1.60). What could have gone wrong? I don´t think it is ethanol, as I did do a dry spin and ethanol should not influence the 260/230 value... Could guanidin hydrochloride (from the Qiagen kit) make the sample float? What else could be the problem?

Thanks a lot!

We work a lot with an enda+/endonuclease+ strain of ecoli (stbl3), so for minipreps we just do the PB wash step and things work fine.

However, we sometimes have trouble with low quality DNA from maxipreps using the Qiagen kit.

I couldn't find this anywhere: Do Qiagen maxi kits tolerate endA+ ecoli strains? I read some other maxi kits have no trouble with endA+ strains, but I couldn't find anything about Qiagen's maxi kit.

I'd like to just switch to another ecoli strain, but my professor doesn't believe anything without extremely definitive evidence.

I need to fragment my DNA around 200-600bp to make ChIP-seq. I used a Sonics Vibra-Cell sonicator with cup horn (750W) 10s ON and 10s OFF (longer pulse allowed by this sonicator). The samples remained on ice during sonication. I can see a smear of 200bp to 1000bp fragments, but some of my DNA remains unsonicated. After sonication I did crosslink reversal DNA purification (including RNAse and Proteinase K treatments). I used about 200ng DNA in the agarose gel (1%).

Note: My samples are tomato and I using EpiQuik Plant ChIP Kit (Epigentek).

I am performing a phenol/chloroform extraction of DNA. Unfortunately, I have to equilibrate the pH of the phenol by myself, and mix it with chloroform afterwards.

The way I did the pH equilibrium is by pouring some TE buffer (pH=8.0) into the phenol and shaking it rigorously for 2 minutes, and letting it equilibrate and phase separate. After that, I measured the pH of the phenol. it is 6.8 instead of being 8.0, which is desired.

Is this correct?

Hello. I generated a few ChIP-seq libraries using NEBNext® ChIP-Seq Library Prep kit. I've never had any problems in the past and always had nice libraries.

Recently, I came across one issue. The libraries still look alright on a DNA HS Bioanalyzer chip but they are really low concentrated (0.1nM) based on qPCR using the KAPA Library Quantification.

We perform a PCR enrichment of adaptor ligated DNA and then we run it on a gel for gel size selection. I've attached the image of one of the library just before gel size selection. 

For troubleshooting purpose, I've also attached a Bioanalyzer profile of one of the library post-size selection and a gel I ran after qPCR quantification. The gel clearly shows how there is massive amount of bands around 120-130bp which corresponds to the adapters length. 

Do you have any clue about what the issue can be? 

Thanks

Federico

Will this change the kinetic energy of the buffer or what?

I'm trialling a recently described salting out protocol to extract HMW DNA from insect tissue: https://www.biorxiv.org/node/716332.external-links.html.

The yield so far is good (~2-6 ug DNA / 30 mg tissue), and my study species is fairly large in any case, so that's not a huge concern. A quick check with a TBE gel seems to indicate that most of the material is >15 kb.

OD values are abysmal. 260/280 sits around 1.4-1.7, 260/230 is even worse from 0.4-0.8. The latter concerns me the more. There's no phenol or glycerol anywhere in my protocol, so that can't be it. I suspected it might be the salt, but I've seen no change after two precipitation/wash cycles with 1/10 vol. sodium acetate + 2.5 vol. ethanol, and resuspension in TE. All OD values measured with a Nanodrop One.

The downstream requirements are 260/230 = 2.0-2.2, 260/280 = 1.8-2.0.

Any recommendations on the source of the contamination? Stray organic debris? I'm grinding my tissue in liquid nitrogen, sometimes the sample (leg muscle) has exoskeleton attached which could introduce wayward polysaccharides.

I wonder if anyone has a positive experience for improving DNA quality following extraction with Chelex? This method is great for improving the yield, particularly for samples low in DNA or material, but it is also known for resulting in low quality DNA. The absorbance spectra are generally poor. Some methods employ proteinase treatment in concert with Chelex, but in our hands this does not improve the absorbance spectra in a remarkable way. Therefore, I would appreciate any suggestions on how to improve quality of DNA extracted with Chelex.

Hello everyone!

I am trying to make a library of metagenomic DNA in the plasmid PBlueScript SK+. I fragmented metagenomic DNA by sonication and I confirmed the average size of the fragments is 3kb. I then repaired the DNA fragments with Klenow fragment (NewEngland #EP0054) so that the the ends are blunt. After that, I purified this reapired DNA. Previously, I digested the Plasmid PBScript with EcoRV that also leaves blunt ends. I tried to performed ligation in several Vector:Insert ratios (1:3, 1:1, 3:1...). None of the ratios gave a large number of clones but Ratio 3:1 exhibited the best results in terms of number of colonies and higher proportion of white/blue colonies, which is good .

I extracted plasmid from 15 white colonies of this ligation, I performed Restriction digestion with enzymes XhoI and NotI (that flank EcoRV at both sides in plasmid PBlueScript) so that I could see two bands in agarose gel (1 Plasmid backbone and 2 metagenomic insert). The problem is I obatined a unique band for most of the clones and, moreover, this single band does not match the size of the empty plasmid. Its smaller !!

To me, this result does not make sense. I thought there could be recombination events in the cell but I´m using DH5 alpha strain so it should not happen. Could it be due to an effect of ligation of so long fragments whit blue ends? Am I missing anything?

I have no experience with library construction so any help will be very helpful.

Thanks a lot in advance!

Jorge

I wish to perform DNA drop dialysis and I wonder if I must use 0.1 um (or smaller) ester cellulose membrane or I can perform it in larger pores such as 0.22 or even on a piece of common tubing membrane with similar cut off?

I am trying to purify a 5.8kb plasmid from agarose gel. I have used 2 different kits and I remain on experiencing very low DNA yield after elution. I have tried to elute in 50uL, 30uL, and even did pooling (showed a better result). I begin with around 1ug of DNA but after purification I end up having 12ng/uL.

Is there any in-house protocol I can use?

I regularly purify cell free DNA from patient plasma samples using Qiamp circulating nucleic acid kit, followed by analysis on BioAnalyzer which indicates genomic DNA contamination in few of the samples. I am looking for the protocols to purify cell free DNA from those samples by size selection. I have been thinking to use Ampure XP beads for the same. If anyone has used the same for similar purpose, it will be very helpful, if you can share your protocols with me.

Or you can suggests any other kit available for size selection of DNA below 200 bp, please let me know.

I digested 2.5 ug of plasmid and PCR product, and after digestion ran all of it on a 0.8% gel.  I excized my bands and proceeded to a gel purification using Thermo Scientific's Gel Extraction Kit.  After gel purification, I ended up with 8-20ng/ul of DNA by spec.  Where is all of my DNA going?  I was going to use these fragments for cloning.

Is the DNA binding chemistry between DNA and column provided with the kit same in case of PCR purification kit and gel extraction kit?

We perform Sanger sequencing for varied range of PCR products. We usually employ ExoSAP for PCR product clean-up reaction. Due to unavoidable reasons we are looking for ExoSAP alternative.

Does ExoCIP from NEB perform equally good?

Technically what is the difference in the enzymatic activity of Calf Intestinal Alkaline Phosphatase and Shrimp Alkaline Phosphatase? Which enzyme has optimum performance for PCR product clean-up activity?

I am linearising plasmids for standards for qPCR. I purified my plasmids using Sigma miniprep and got the following results:

Plasmid 1 (P1) = 277.2ng/ul and 260/280 = 1.87

Plasmid 2 (P2) = 93.8ng/ul and 260/280 = 2.02

I have linearised them with Sph1 whose restriction site is not in the desired insert sequence. And followed the manufacturers protocol for the digestion.

Then I have run them on a gel, but no bands at all are visible. I am loading 200ng of plasmid onto the gel against 200ng of uncut plasmid to compare for linearisation. What could cause no bands?

After this I usually purify the linear plasmid. Using QIAquick purification kit from Qiagen. But most of my results end up <10ng/ul and very impure.

Any suggestions why?

I need a huge concentration of restricted product. So can i just digest them in a 50 uL reaction with high volume of enzyme (say 5-7uL). This is just to run the digested product in a single gel and do a single shot gel elution.

Thanks in advance

Hello,

I am amplifying a number of amplicons >1000bp from various plasmids and the PCR goes well.

However after a purification procedure (Qiagen's colomn method) a strange new band appears for all my amplicons, regardless of the template DNA.

I am very confused by what these could be and have done many controls to try to stop them from appearing (new buffers, new water, no vortexxing of solutions)

I am hoping that someone may have had a similar experience or perhaps someone may give some insight into what these may be and what it takes to stop them from appearing.

After some consulting I decided to use a commercial pcr cleanup spin column kit to concentrate DNA from my CHIP for sequencing. The DNA was in elutate of sodium bicarbonate and 1% SDS. Instructions required me to dilute 6x in binding buffer, so this meant 9mL per sample column. When combined, precipitate came out, I assume this was the SDS. All of the protocols I've seen that use sodium biocarbonate and SDS to elute out dna jumped straight to purification using p/c or kits so I just moved on.

I ran the liquid through the column and near the end when I was washing them I noticed that the membrane of the tubes looked a bit crumpled and the wash buffer was dripping through it somewhat faster than in control columns and whitish precipitate was in the wash. I tried looking at the membrane at every angle but I didn't see any hole. After I eluted I noticed a tiny pellet of whitish stuff at the bottom of the tube. I'm not sure whether this was SDS or silica or what. I had the elutant tested through qubit and the samples showed very low to below threshold amounts of DNA. But this was CHIP pulldowns so this is not necessarily indicative that something is wrong. I was wondering what sort of steps I should take from here. Should just move on and see if I can create a library and do a PCR check or assume the columns were broken and try to reprocess the binding buffer waste which I kept? Multiple companies I contacted seemed to think that the spin columns should be able to withstand that volume although one of them (Thermofisher) apparently has columns with membranes that look thicker than the kit I was using.

According to protocols I found from Nature (Heckman et al 2007) and Cold Springs Harbour (Forloni et al 2018), the PCR products must be purified using gel electrophoresis and then a QIAquick gel extraction kit or a similar product. According to a senior person in my lab, we usually use ethanol precipitation for PCR products because the gel method results in loss of a lot of the product. Is this a question of quality over quantity?

Also, is there a difference between the QIAquick gel extraction kit and the QIAquick PCR Purification kit, and which is better?

I have amplified my samples using ITS1F and ITS4 primers. The gel has a faint band under the main band, the PCR settings are right as they work on other samples. The faint band is the same as the main band as I have sent them both for sequencing.

I did a PCR, then did a 1:10 dilution and did another PCR on the dilution using 1 ul of diluted product and 0.5 ul of diluted product in 50 ul reaction. My supervisor says I should mix both reactions together, then do something to purify the samples so I only get the main band to send off for sequencing. It doesn't involve a kit or running any more PCR, apparently it's really simple!

Does anyone have any suggestions?

I face many difficulties in gel purifying the restricted products

So is there any other way to ensure cleanup of the digested products without running them on gel?

I start with impure PCR products of >500ng/ul, 260/280=1, but peaks present at 260, and end up with peaks near 230, about 10ng/ul concentrations of 260/280=1.5 or so, which I assume is not DNA since there is no peak at 260. Is this due to contamination by guanidinium salts from the buffers? Or ethanol? What can I do? Gel electrophoresis with a 2% agarose gel for 25 min at 70V showed DNA of the desired sizes, though not necessarily large amounts, and after purification, there was pretty much nothing.