ResearchGate Q&A lets scientists and researchers exchange questions and answers relating to their research expertise, including areas such as techniques and methodologies.
Browse by research topic to find out what others in your field are discussing.
PCR succeeded with Taq but failed with KOD?
I succeeded with PCR from genomic DNA using Taq. But I need to sequence the PCR product, so I want to use KOD instead of Taq. However, I cannot get any band using KOD, I tried different annealing temperatures but all failed, what else could I try?
Thank you very much!
Have you decreased the extension temperature from 72°C by 68°C? or changing the Mg concentration? Could work.Following
What is the physiological effect on plants if the soil is contaminated by a small, or large amount of cyanide?
I am curious about the physiological effect of a plant if the soil is contaminated with cyanide.
Can soil microorganisms converts the cyanogenic glycosides in soil to hydrogen cyanide which can evaporate to the air?Following
What are tropophyllous (tropophilous) species, examples?
There are species called 'evergreen' and 'deciduous'. Some species are labelled as tropophyllous (tropophilous) species and considered between evergreen and deciduous. Is that so?Following
Does percent of crystallinity affect the adsorption of MWCNTs towards the contaminants?
I treated my MWCNTs, and it showed that the percent of crystallinity increases by the XRD analysis. The MWCNTs then will further use for the adsorption of contaminants. I just want to know, does the adsorption capacity of MWCNTs can be affected by the percent of crystallinity of MWCNT? If it does, how does actually the crystallinity can affect the adsorption? Does it will increase or decrease the rate of adsorption?
I'm also glad if someone can attach any literature review regarding these.Following
How can I standardize maximum lifespans calculated from transition matrices that differ in their time-interval?
I am comparing longevity using as a proxy simulations of maximum conditional lifespan based on published matrix models. Most of the transitions that I’m using are based on annual population changes (from 1 year to the next year), however, some other transition matrices are based on variable time intervals (months, 2-year, 5-year, etc… How should I standardize these values to make them comparable? An easy way I have in mind would be just to divide the estimated lifespan by 12 in the case of monthly matrices or multiply by 2 when dealing with lifespan calculated based on biannual matrices, but I am not sure if that would be appropriated. Any suggestion?Following
Can anyone explain what is the relationship between binding energy and stability in docking?
I would like to know the relationship between binding energy of ligand and protein in docking and the stability of the conformer.
Thank you sirFollowing
Can we expect well resolved peaks in a blank injection (methano: water) in a gradient program?
I am trying to develop a gradient method for metabolomics study. I programmed a gradient and tries to run blank injections containing methanol : water in the ratio of 1:1 using the LC-QToF (MSe scan). To my surprise the TIC doesn't show any peaks but the low energy BPI chromatogram showed number of peaks. I tried washing the column and changing the mobile phase, changing the sample vial. etc but in vain. Can anyone suggest me more troubling shooting. I would appreciate if anyone could let me know whether they too observe similar phenomenon.Following
What is the procedure to mix nanoparticles as filler in to the glass fiber and polymer resin matrix?
I want to use nano particles as filler in GFRP.. Many researcher used to mix the nano particles into resin by stirring arrangement and changing the temperature of resin while mixing..
Nanoparticles can be best incorporated to a polymer matrix by melt blending technique. I think you better choose an internal mixer (like Brabender) for better dispersion. The method may also depend on the nature of the filler and polymer in your study.Anyway you have to adopt the technique which gives maximum dispersion so that your material possess enhanced properties.
What is the biological aim for each step of histone extraction from brain tissue ?
We are currently working on histone extraction from brain tissue, However, I need to know the concept of each step! from where could I get it ? or If could you provide me with it here?
For tissues (treated and untreated), weigh the sample and cut the sample into small pieces (1-2 mm3) with a scalpel or scissors. Transfer tissue pieces to a Dounce homogenizer. Add TEB buffer (PBS containing 0.5% Triton X 100, 2 mM PMSF and 0.02% NaN3) at 1 ml per 200 mg of tissue, and disaggregate tissue pieces by 50-60 strokes. Transfer homogenized mixture to a 15 ml conical tube and centrifuge at 3000 rpm for 5 minutes at 4°C. If total mixture volume is less than 2 ml, transfer mixture to a 2 ml vial and centrifuge at 10,000 rpm for 1 minute at 4°C. Remove supernatant.
Resuspend cell/tissue pellet in 3 volumes (approx. 200 µl/ 200 mg tissues) of extraction buffer (0.5N HCl + 10% glycerol) and incubate on ice for 30 minutes.
Centrifuge at 12,000 rpm for 5 minutes at 4°C and remove the supernatant fraction to a new vial.
Add 8 volumes (approx. 0.6 ml/ 200 mg tissues) of acetone and leave at –20°C overnight.
Centrifuge at 12,000 rpm for 5 minutes and air-dry the pellet. Dissolve the pellet in distilled water (30-50 µl/ 200 mg tissues).
Aliquot the extract and store the extract at –20°C or –80°C.
If there is residual acetone, the pellet will be hard to dissolve. Remember the reason for dissolution: H2O molecules form extensive H-bonds with participating side-chain moieties on proteins. In presence of acetone (which is an organic solvent) hydrophilic moieties would not be exposed adequately, with non-polar surfaces being exposed. Therefore, histones will not dissolve properly if there is residual acetone. This is analogous to trying to dissolve a DNA pellet with, say, 20% ethanol.
Here is a caution, though! Over-drying makes it harder to dissolve the pellet. Under-drying or over-drying cause problems ONLY when there is substantial contamination from other proteins or DNA. If your preparation is uncontaminated, you should not face any difficulty at all.
You can dissolve the histone pellet in whatever volume of water you want. The only constraint is the histone concentration. It should not so dilute that you cannot take sufficient amount of histones in your down-stream reactions. And your experience is true: solubility is increased by increasing the solvent volume.
Later adjustment of the protein concentration should be in water. You can have 0.2mM PMSF (to prevent proteolysis), while some also recommend 15mM 2-mercaptoethanol (to keep the two H3 molecules from forming a disulfide linkage). Once the histone is all dissolved, you can spike Tris (pH 7.5-8.0) to final 10-20mM if required, but not necessary. In special cases, one may need to store histones in octameric form. That requires some special procedures.Following
How do I transform negative emotion, i.e. fight or flight, in terms of valence and arousal?
Negative emotion is usually referred as "fight or flight" state. Within 1 to 10 (negative to positive) for valence and arousal, where is the location of this negative emotion?
Everyone is familiar with emotion, feelings, affect and believes they understand it. But there is a big difference between familiarity and understanding and most people can only define even their own emotions in a circular fashion. The history of psychology if full of failures in the measurement of emotions. The mind (and feelings) is a black box which we cannot see into, even with psychological tests. Tests of aptitude and achievement tend to be very reliable and valid and useful. But psychological test on feelings and emotions are not. The real progress in clinical psychology has come from measurement focused on concrete behaviors. B. F. Skinner's work and his schedules of reinforcement is still used today for controlling human behavior. For example, casinos and the best uses of behavior modification make use of schedules of reinforcement. Best of luck in your work on emotions!Following
Looking for technique to study protein changes in tissue??
Hi, I have to find out six proteins change due aging in muscle tissue. I am performing western blot right now. Is there another technique available to get accurate protein concentration? I don't have purified proteins to use as standers so can't perform ELISA.Following
Anybody who has licensed installer of STELLA (model maker) and VENSIM?
This software are very good for analyzing ecosystem complexity. I hope somebody could help me wth this.Following
What is in your opinion the proper agarose% - volts/cm for separation of aprox. 1500bp in gel electrophoresis?
I want to separate relatively close bands (difference of aprox. 50bp) for isolating each single band by cutting them from the gel to send them to sequence. Any advice would be appreciated.
A PAGE gel will be better to call such small differences with confidence.Following
What do you think about this assumption: 'The bigger it is the final size of the bivalves the shorter is the distance between VO2 and CR exponents'?
I measured the respiration rate and clearance rate of geoduck clam to determine their allometric coefficients. Since the final size of geoduck clam is nearly 1 kg, the comparison between species might be lame. If you have determined the allometric coefficients of RR and CR for a bivalve species which is similar size to mine, please let me know. Many thanks.
Thank you very much for your opinion and references. Although I did not measure the gill surface area, I'll try to compare two coefficients in relation to the mass between species again. Since geoduck has huge amount of tissue compared to the shell weight and it lives for up to 160 years, I could not get my head around those coefficients.
What are the types of medication error?
Hello, I,m try to discuss all types of medication error to try decreasing these errors as possible.
US FDA also has a page with links to resources on medication errors and patient safety:
Institute of Medicine produced a report on prevention of medication errors, you can either access it online or download pdf (you need to register to do so, but I believe it it free):
I hope it helps.Following
Where to get human corneal endothelial cell (HCEC) line?
HCEC are difficult to expand ex vivo. A few research groups have succeeded in immortalizing HCEC, but is there a commercial source?
The question was asked long ago but I'll still add the information as it may help others who are interested in this field. The corneal endothelial cell line (B4G12) is commercially available from DSMZ: http://www.dsmz.de/catalogues/details/culture/ACC-647.html
However, it is important to note that the immortalized corneal endothelial cells are only good as a model cell type to understand different functions of corneal endothelial cells. However, these cells can not be used as a cell therapy or for tissue engineering approach for actual corneal patients. The "unlimited" proliferation cold potentially cover the trabecular meshwork of the eye. We have worked with both cell types and we think that the primary cells separated from donor corneas cold be the best cell source for cornea therapy.Following
Is it mandatory to have the value of Cronbach's alpha above .70?
In my study using questionnaire survey, I have four variables and job satisfaction is one independent variable. I measured job satisfaction with a 3-item scale. Responses were captured in a 5-point LIkert scale. The value of Cronbach's alpha is .65. However, the value of Cronbach's alpha for other variables (captured with 5-point Likert scale are above .70. Can I use the data (with job satisfaction having alpha value below .70) to test my hypotheses?Following
Where can I easily get Pseudomonas aeruginosa?
Hello everyone. Where do you think I could easily get Pseudomonas aeruginosa from the environment? I used to easily culture Vibrio parahaemolyticus from different shellfish on Vibrio chromogenic agar previously. Anybody with experience on Pseudomonas aeruginosa?
The bacterium would not be found in air in our two hospitals. The organism likes water. Anywhere there is standing water the bacterium might be isolated.Following
Does anyone have a copy of Lewinsohn et al (1993) on adolescent psychopathology?
Which is the easiest anti-cancer drug to load into PLGA nanoparticles?
Could anyone suggest from their experience with PLGA nanoparticle preparation as to which is the easiest and most efficient anti-cancer drug to use as a model? I plan to use PLGA mw 55-79KDa, nanoprecipitation method to load the drug followed by amicon filtration.
Hi Sudip and Marco,
Thank you for your inputs. I have been trying to load paclitaxel following the protocol of some very good labs but unable to get a clean synthesis (the drug even at 0.1% initial loading crashes out with both dialysis and nano-ppt methods). I am suspecting the viscosity of PLGA i'm using is too high (59-75 KDa, ester terminated) I think I will try doxorubicin next.Following
3D Printing vs Traditional Manufacturing?
In your view what are the main differences between 3D printing and traditional manufacturing?
What are the advantages & disadvantages of 3D Printing & Traditional Manufacturing?
Do you think 3D Printing will cause Traditional Manufacturing to collapse?
I do agreed , this is a revolutionary invention. 3D prototyping has taking place in China. Itshelp to reduce the cost of prototyping , and design cost . In short term , the impact is very minimum.Following
(continuous)Can anyone identify this fungus?
This is what fungus? I first saw the colony back, think is rare, don't know, hope to know advice. Thank you!
Morphological identification of opinion, I think the fungus may be Paecilomyces, but the current species name is not known. can You hint me what fungus? please help me mark features in the diagram, all right? Thank you!Following
Which technology file/ transisitor to be used at RF frequency?
I want to implement a CMOS inverter that can work at GSM band (850MHz/900MHz) in SPICE tool. I am using TSMC's 180nm model file. I am not getting exact output at this much high frequency. So, can anyone suggest which model file to be used for higher frequencies and from where can I get it?
Please answer ASAP.
Can anyone suggest which CAD tool is suitable for implementation of RF front end design?Following
What test can be used to assess anxiety, depression and anger in children 8 to 10 years?
I would like to know what test can be used to assess anxiety, depression and anger in children 8 to 10 years. We are conducting a study on the emotional wellbeing in children with oncological diseases and we need to evaluate these states. Test such as IDAREN, IDEREN, Kovac, are for teensWe are conducting a study on the emotional wellbeing in children with oncological diseases and we need to evaluate these states. Test such as IDAREN, IDEREN, Kovac, are for teens
Anxiety is a feeling which is measured almost always by self-report. There are many anxiety scales with good psychometric qualities you might use. Of all the scales I have used, the most useful is the Children's Depression Inventory 2nd edition. Children respond well to the child-focused wording of the questions. Children think and feel out their answers more than on the usual true false formats.Following
How do I design or choose primers for porcine mesenchymal stem cell targets?
I'm trying to do a gene expression study for porcine mesenchymal stem cells which have been induced towards differentiation using different chemical agents.
At first, I tried to design my own primers by using "consensus sequences" I found in this database for the pig genome (http://pig.genomics.org.cn/transview.jsp?transcript=ENST00000230354&seq_type=all). I would get the consensus sequence and use Primer 3 to design a primer pair for that sequence. Then I used UCSC In-Silico PCR to check the specificity of the primers. Before using UCSC In silico, I tried using Primer Blast but it confused me as even published primers did not seem to be specific when I used Primer Blast..sometimes even declaring there were no relevant matches or sometimes giving so many matches none of which contain the expected amplicon. So anyway, using In-Silico and the pig's reference genome (2011) they have there, I made sure the primers I designed were specific to one product only.
The problem is when I ran my primers, I got a signal in my "no reverse transcriptase" controls that have the same peak at the melting curve as my expected amplicon. I thought this was a contamination problem so I bought new primers and took care in diluting them and I also bought new primers from a published paper. Interestingly, my fresh primer (which I have designed) still gave the similar peak for my "No RT" control, but the published primers didn't give a peak in their "No RT" control. I do not understand if this is an inherent problem in my primer design as it doesn't seem to be a primer dimer because the peak is the same as for my reaction tubes. I am testing for housekeeping genes right now. I am so tired already from designing and redesigning primers and doing standard calibration plates for so many times already that I have resigned to just using the published primers. However what worries me is how do I then choose primers for the specific cardiac genes I want to study. If I attempt to design them again similarly as to what I described earlier, then I might face the same issue in the signal in my no RT control. I have ruled out the problem of contamination as my "water and primers only" control doesn't give any signal. There are not much published primers for the porcine cardiac genes that I want. There are a lot of studies however for human, mouse and rat. Can I simply use these?
Thank you Michaela for the input. That is also why I decided to just design my primers as well. I did run a gradient. Thank you for the well wishes, I hope I figure this one out soon!
Hi Lisa, I've been coming across that advice about designing primers that cross an intron, but I don't know how to properly do that. You see I'm using this "consensus sequences" I got from a pig genomic database (that I mentioned above in my question), and I believe what they have there are the coding regions only. If I include an intron, will it still be possible to keep amplicon sizes between 80 to 200 (as advised by my BioRad SYBR Green Kit)? I was trying to look for a website that describes a method in a step by step manner but couldn't find any. I have no background on biotechnology and all of this is new.
Hi Michael, thank you so much for the detailed suggestions. I did run my PCR products on an agarose gel and they are of the same size of the expected amplicon. I have attached a picture of the two gels. The first one is when I did using only the primers I designed wherein everything had a peak in the no RTC control. It was my first time to do PCR that time and so I don't think it was due to any contamination of post-PCR products. The second one is when I just used the ACTB I newly designed (this is a different ACTB primer design from the previous one), along with HPRT1 (Nygard 2007) and SADH (Nygard 2007). Again there is a signal for my ACTB No RT controls however for both HPRT1 and SADH there are no signals. The lanes with "?" on top were reaction tubes that I think had primer dimer problems because they had two peaks so I checked them.
Thank you so much for all your help! I really appreciate it.Following
What is the UV cut off wavelength in benzyl alcohol solvent? and how find theoretical?.
what is the UV cut off wavelength in benzyl alcohol solvent? and how find theoretical?.Following
Can anyone help learning dissipative particle dynamics (DPD)?
I am looking for some references to learn about Dissipative Particle Dynamics (DPD). Any reading material, suggestion regarding DPD codes would be really helpful.
Thank you very much.Following
Are there any management articles that use the term magical realism?
I am interested in tracking down either empirical or theoretical papers.
Excellent. Thanks Oluwole!Following
Could the rural tourism strategy in Uttarakhand be helpful for developing a sustainable economy in rural Uttarakhand?
Role of rural tourism in Uttarakhand.Following
Can anyone tell me what the molecular radius of TiO2 is?
Thanks in advance for your replies.
I find the radius in two ways. ( i) lattice parameters (0.3793*0.9502=0.36nm2 for anatase) (ii) bond lengths (0.198*0.195= 0.038 nm2 for anatase) . I am not sure which one is correct.Following