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  • Paulo Paiva added an answer in Performance Evaluation:
    How to measure subjective skills for surgical team performance evaluation?

    Someone how knows if there's specialized guidelines for defining ways of measuring performance of surgical teams in a standarlized way?

    Paulo Paiva · LabTEVE

    Thank you H Gin Chong for help. I've found some interesting works of yours, I'll read them and sent you some inline answer.  

  • Neeraj Misra added an answer in Gaussian (Software):
    Anyone have idea which institute helps for free use of gaussian software?

    pls. send me its contact

    Neeraj Misra · University of Lucknow

    You can also try "gamess" 

  • Darryl D Siemer added an answer in Electrochemical Impedance Spectroscopy:
    How can I measure the impedance of stainless steel electrodes of a device implanted under the skin?

    I would like to measure the impedance of stainless steel electrodes of a device implanted under the skin. So, the working electrode is each of the stainless steel electrodes and the solution is the body (in vivo).

    What metal and area should be used for the reference and counter electrodes?

    a) Is it true that the area of the reference electrode doesn't matter because ideally no current flows between the working and reference electrode? Similarly, how much bigger should the counter electrode area be relative to the working electrode?

    b) I've read that a non-polarizable electrode should be used as the reference electrode, so it should be something like Ag/AgCl, right? What about counter electrode?

    Also, is it true that the reference electrode should be close to the working electrode, while the counter electrode should be far away? We are flexible with placing these electrodes anywhere in the body.

    Darryl D Siemer · Idaho State University

    This is a tough question to answer without a better description of what you are trying to  accomplish.  If the object is to measure net current at a working electrode as its electrochemical potential  is changed (swept), you will probably need some sort of genuine reference electrode along with a counter electrode.  Stainless steel is not a reliable reference electrode because its absolute potential depends upon what's in the sample solution (bodily fluid) which may be neither  known nor constant. The size, position, and/or composition of the auxiliary electrode are not  important as long as it's not too close to the other electrodes and made of a good conductor which isn't corroded by  bodily fluids (stainless steel is usually OK).  If the reference electrode (eg, Ag/AgCl/Cl-)is relatively large with respect to the working electrode and fitted with a low impedence junction (e.g., a not=too-small i.d.  glass tube plugged with a 1 M KCl  saturated "thirsty glass" frit) whose frit/sample end is situated  close to the working electrode,  it can serve double duty as both the reference & and auxiliary electrodes.

  • Sudev Naduvath added an answer in Radiation:
    Does radiofrequency radiation cause cancer?

    Does radiofrequency radiation cause cancer?

    Sudev Naduvath · Vidya Academy of Science & Technology

    That is a good question of public interest. It widely believed that uses of cell phones result in serious diseases like cancer.  People believe that the radiation is dangerous to the well being of the living things.  We usually see many public protests against establishing new mobile phone towers in the civilian areas. Not much significant studies in this matter have reached to the public also. It is the case for all types of wireless communication networks. Certainly, as a layman, I am very much interested to know the expert opinions and I expect more expert answers and serious discussions in this matter. 

    Thank you for this question and let us hope the answers will be helpful to reduce our anxiety level.

  • Vaibhav Mantri asked a question in Algae Culture:
    Are there any uses of algal photobioreactor in macroalgae culture?

    Although there are several studies pertaining to use of algal photobioreactor in micro algae culture, I would be happy to know if similar studies are being carried out in macroalgae (seaweeds)? It would be good, if I get few of the recent references.  

  • Phil Faidley added an answer in Fires:
    Have you conducted empirical research about juvenile fire setting?

    I am conducting an analysis of records of intervention with juvenile fire setters, searching for correlates between the identified risk factors (social, environmental, behavioural, psychological) in the young person, and the methodologies used to set fires - the versatility of the fire setting.

    I have not found much research that has been conducted to date that links these artefacts explicitly. 


    Are you aware of research that explores the interconnection between risk factors and methodologies? 

    Phil Faidley · New Zealand Fire Service

    Thank you very much Abigail.  I have downloaded your articles and will be interested to read them.

    Kind regards

  • A. Subba Rao added an answer in Soil Analysis:
    Which is the most effective method of diagnosis of nutrient constraints in perennials?

    In perennial crops , identifying nutrient constraints is truly  a complex exercise. An entire range of diagnostic techniques are being used , starting from soil analysis , tissue analysis ( Using index plant parts ) , metallo-enzymes , deficiency symptoms as  morphological descriptors, juice analysis, trunk injection to floral analysis,  of late. None of them alone is sufficient  to identify the nutrient constraint precisely matching to field conditions. Every method has its own merits and demerits. With the result , it is  often questioned that the currently available methods of diagnosis of nutrient constraints , are more inclined to diagnosis for the next season , overlooking the problems in the current season  standing  crop . This kind of constraint in diagnosis is so rampant in perennial crops. I wanted to know from researchers across , how to solve this so complicated issue?.

    A. Subba Rao · Coromandel International

    Dr.Srivastava,we can do any  newer aspect for  research purpose  but we can not ignore practical agriculture or horticulture.To my knowledge there is no better tool  than foliar diagnosis of perennial plants at appropriate stage in the  season and index tissue.If right instrument (Dr.Laing) is used  and prompt service is provided Dr.Coutinho),plant analysis provides right data for fertilization,especially for correcting micro nutrient deficiencies through foliar sprays.Progressive farmers may be applying  major nutrients on their own  and their schedules can be corrected during the season  or next season.It is also believed that the the nutrients   take sufficiently long time to reach the active roots in the soil. So one season delay in perennial crops may not be a big  problem.Soil analysis helps  to serve two purposes in tree crops-one during planting and establishing and second for monitoring excess accumulation of nutrients in crops like grapes.This can also be appropriately be used in horticulture.

  • Arezoo Dadrasnia asked a question in Bioprocess:
    I am look for innovative bioprocess in metal sorption, any suggestion?

    Regarding the metal biosorption from wastewater, what process is currently novel and better economically and environmentally..

  • H Chris Ransford added an answer in Superstitions:
    Is there room for religious thoughts in a scientific mind?

    Two of my previous questions were on the influence of emotions and/or  of superstition, on a scientific mind.

    Many of the answers we got were absolutely surprisingly unique and brilliant.

    I thought that we should go on, towards thr definition of a m,ore complete scientific mind. 

    Should scientists only use their pragmatism in problem solving, or can they use their intelligence in broader views of the World?

    H Chris Ransford · Karlsruhe Institute of Technology

    It would depend on what you mean by 'religious'.

    If by religious you in fact mean spiritual - i.e., the inchoate possibility that the universe or metaverse may host something transcendent and infinite that we are never in a position to fully grasp (because of the phenomenon of emergence, and also because of the total impossibility to significantly understand, apprehend and/or map infinities from a finite base), then the answer to your question is certainly yes.

    If by religious you however indeed mean religious - i.e. the embracing of any particular choice of arbitrarily structured, mutually contradictory human-made religions with their trail of peremptory say-so's, petty rules, illogical leaps of faith, meaningless rites, pointless costumes, asserted truths, and so on, then the answer has to be a resounding no.

  • Timothy A Ebert added an answer in Pesticides:
    Does anyone have dissipation curves fo pesticides in soybean, in terms of insect mortality?

    I need bibliography, data to fit or already fitted curves of pesticide dissipation. I'm looking for the decrease rate of insect mortality in crops after spraying; for example, novaluron, cypermethrina and chlorpyriphos on soybean.

    Timothy A Ebert · University of Florida

    I think you are looking for something that does not exist in a form that will be useful to you. A degradation rate calculated by someone in Ohio (USA) will have little relevance to Argentina. The degradation curves will be influenced by:

    1) Season

    2) Humidity

    3) Rainfall

    4) Formulation

    5) Adjuvants

    6) Application technique (fogger, hydraulic, spinning disk, etc...)

    7) There will be an interaction between the target insect behavior and application method.

    8) Species, genotype, and phenotype of the target insect

    9) and the list goes on. If what you want are published articles to help you design experiments, then we might be able to help. However, doing a literature search through the University library should work well. Also Google Scholar is useful.

  • Andrei Gonzales I. added an answer in Two-way ANOVA:
    Which ANOVA Test is Better for my data?

    Hello to everyone.

    First I want to say that I checked other similar questions uploaded here but I am asking because I still don't know for sure whether I am doing the right statistical test for my data.

    Here is an imaginary example (check the attached picture). Lets say I have a T cell suspension, samples of which I treat with different concentrations (A, B, C, D) of a given drug. I have a total of 3 subsets of samples (one set of cells that are non activated, a set of anti-CD3 activated cells, and a third one activated with PMA+Ionomycin). The role of controls is served by untreated with the drug cells (denoted as "Cells only" on the figure).

    Let me note that I am using GraphPad Prism to make graphs and do analysis. Therefore, if I want to perform a statistical analysis of the data above I should use Two-way ANOVA test with Boniferroni's post test, right? I come to this conclusion since both the activation of cells (anti-CD3 or PMA/Ion) and the drug used for treating the samples may affect the output. Am I correct?

    Moreover, if I build new graphs for each of the subsets (Non-activated, anti-CD3 activated, and PMA/Ion activated) and use One-way ANOVA with Boniferroni's or Dunnet's post tests instead and compare the results for "Cells only" with the results for concentrations A-D, then this analyzing will not be correct due to the fact in this way I will omit the effect of the activation of cells and just consider the effect of the drug. Am I correct on this one? 

    Thank you in advance.

    Andrei Gonzales I. · Universidad Mayor de San Andres

    First step, between which groups you want to see the significance? ¿which is the question of your experiment?, what is the value of your N ?, second, Your data play a normal distribution?.

    Perhaps you can use a two-factor ANOVA and to see exactly where the significance occur you can use the Tukey HSD (Honestly Significant Difference)

    If your data do not meet the normal distribution can be used the Kruskal-Wallis and Tukey used instead of U of Mann Whitney But we test between the groups in pairs, 1st with 2nd; 1st with 3rd and 2nd with 3rd; In this way we can know which of these is given the difference.

  • Lillian Onwuha-Ekpete added an answer in Cytometry:
    Can I change the parameters for forward and Side scatter for my experiment in nflow cytometry after compensation?

    Hello all;

    I am examining bovine lymphocytes and I am staining them with 4 colors for flow cytometry. I have done the compensation controls and they are compensated now. When I run the samples for my actual experiment, the population look different from that at the compensation and I need to change the parameters, can I and will my samples still be compensated?

    Lillian Onwuha-Ekpete · Florida Atlantic University

    Once the voltage is set for that experiment , it can't be changed . That's one of the importance of flow cytometry , it's hard to manipulate data. Changing voltage can alter results dramatically, giving the illusion of something being brighter than it really is or vice versa. You can rerun your samples if they are fixed or next time set the voltage not just on the FSC and SSC parameters of your unstained population but also on your compensation controls.

  • Muhammad Khattab asked a question in Pearson's Correlation:
    In solvatochromism study of UV-Vis spectra, what is the difference between using pearson's correlation coefficient and coefficient of determination ?

    which one of pearson's correlation coefficient and coefficient of determination is more reliable and important. please, give me the meaning with examples.

  • Yong Guk Kang added an answer in Equipment:
    Anyone know of a good source for 100% light-blocking covers that could be made to spec to cover benchtop instrumentation?

    Lovely as the lab is all done up in foil... I'm looking for a place that could make sturdy blackout covers for benchtop liquid handling equipment from photos and specs.

    Yong Guk Kang · Korea University

    although I didn't measure its efficient, 

    For my case, I'm using combinations of Al foil+blackout curtain fabrics.

  • Lillian Onwuha-Ekpete added an answer in Antigen:
    How do you determine the surface density/expression of your antigen of interest to create a panel for flow cytometry ?

    I am trying to develop a multi-color panel  and to do so I have read that I have to match bright flurophores to dim antigens. But, I am having trouble assessing what is high or dim expression. Some people recommend looking into literature; but literature can be vague?

    Lillian Onwuha-Ekpete · Florida Atlantic University

    Thank you

    Mahima, I did figure out how to use the histograms. It helped me see how much separation I would get between my positive and negative population based on a given conjugate.

    Erin, I have started titrating my antibodies and then I'll test out the panel to see how well it works for me.

  • Rachel M. Olinger added an answer in Student Development:
    How do you manage attendance of students?


    I would like to ask how do you provide attendance to your students. I mean what strategies you use in order to require students to attend classes. If I am not wrong, most of the students are generally not interested in attending classes due to many reasons (like most are just interested in getting grades, or they attend due to friends etc).

    In such cases, how do you make sure that students would attend classes regularly and take interest in classes, despite the fact that classes are interesting and engaging. Are there some "student variables" which are difficult to manage at all costs?

    Rachel M. Olinger · Louisiana State University

    I am newer to teaching, at least as far as being the instructor of record, but I have found a few different strategies that have seemed to work so far. I am really passionate about teaching and it's very important to me that students are learning things that will be relevant to their lives or careers. So making the course lecture interesting and relevant to my student's lives is a priority and it seems to really help with attendance. So far, I've gotten great feedback on anonymous feedback tools I've had my students complete and many of them mention how taking the time to learn their names and ask questions about what they want out of the course has made a difference.

    I try my best to incorporate discussion and applied activities into every class, so that it's not just lecture and therefore more intrinsically motivating for them to attend. Now, obviously this means extra prep time, and it doesn't get all students to attend, but I find that I myself enjoy the classes more when I do it this way. WIth a large class it can be tough to do activities, but I find that even with large groups, it's easy to do a partner activity or small group discussions and have members from the groups share what they've discussed. It's still possible to make a large class inviting!

    Also, I have made participation a part of their grade, so those who do not come to class automatically have their grades impacted. My university uses Turning Point software and clickers, so I have incorporated those into my class and it's been great. I use the clickers to have students answer various types of questions throughout the class period. Some of them help me gauge student understanding of topics or engagement in the lecture. Other times I have used clickers to gauge the opinions of the class on controversial or opinion-related topics relevant to the lecture. This helps the class see that there are differing opinions and helps me to see where the majority of my students are in regards to these topics.

    Also, myself and a colleague have used candy as an incentive to participate during the first few weeks of class. I'll bring a bag of Kit-Kats or something and toss them to those who respond to questions or are actively participating. You'd be surprised how many join in after they see this!

    Lastly, and I think most successfully, is I've created a culture of respect in my class. I make it clear to them that I want them to be successful and that I will do my part. But I also make it clear that I expect them to do theirs. That if we want the class to be as good as it can be, we both need to do what's required of us. I encourage coming to my office hours, and find that students are more likely to come to me to tell me about those "student variables" when they impact their attendance if I show them I genuinely care.

    Think back to classes you took in college - which ones were best? Why? Which ones were worst? I try to emulate the classes that stuck with me and avoid doing things that I hated as a student. It's a lot of work, and obviously every semester will be different with differences students, but I really believe these steps have helped and have paid off in my own enjoyment of teaching. Hope this helps at least a little!

  • Narasim Ramesh added an answer in Sensors:
    How could be minimize the aliasing effect due to the downsampling from a signal?

    Suppose that you have a sensor, that you can't change the sample rate or any other feature including the anti-aliasing before the signal be sampled.
    Beside this, suppose that, the control loop of the system runs with a slower rate than the sensor, and you cannot change the control loop rate, as well.
    Now, assume that the sensor rate is multiple of the control loop rate.
    Then, suppose that, after 5 samples of the sensor, the control loop runs once using the latest sample from the sensor (observation: the 5 five samples are stored).
    As far I understand this characterized the down-sampling of the signal, and it produces the aliasing effects over the signal.
    Then, how this effect can be minimized? Should I have to put a digital filter before the control loop?

    Narasim Ramesh · Sri Jagadguru Chandrasekaranathaswamiji Institute of Technology

    @Hugh Lachlan Kennedy 

    Your suggestion seems very interesting and unusual. .Would you care to give some hints/ref /simple example on the design method?



  • Yong Guk Kang asked a question in Second Harmonic Generation:
    Anyone know of practical way to estimate the optical Second Harmonic Generation and Point Spread function generation by computer simulation?


    I want to estimate the SHG occurence from specific patterned tissues or something. (e.g. striated pattern or crystals)

    Is there any practical ways to done these jobs with computer?


  • Sarah Shahmoradian added an answer in Protein Aggregation:
    Will Thioflavin T assay bind to agarose gels?

    I want to look at protein aggregation in a hydrogel but Thioflavin T binds to protein aggregates.  This means I can't use collagen.  I want to find out if agarose, hylaurionic acid or other hyrdogels such as puramatrix or matrigel will react with the assay preventing me from looking at the beta amyloid aggregation.

  • Hafiz Muhammad Yar asked a question in Quantum Dots:
    ZnO Quantum Dots for Solar Cells

    1,Octadecylamine.........2..octadecene ...........I want to make ZnO Quantum Dots for Solar Cells, so i read the Article A study of photoluminescence properties and performance improvement of Cd-doped ZnO quantum dots prepared by the sol–gel method........in this article why they use 1,Octadecylamine.........2..octadecene for what purpose except of these compound what other i acn use? and washed rapidly means ? what method to wash Please if some one guide and help me in this regard.

  • Tom Masi added an answer in RIPA Buffer:
    Does anybody know which protein quantification kit is most tolerant to RIPA buffer while maintaining high sensitivity?

    What are people experiences with protein quantitation methods and the interference of detergents?

    I am trying to quantify the amount of a membrane bound tetraspanin. As such I am using RIPA buffer as (from what I understand) it is one of the best for solubilising membrane proteins and lipid protein complexes.

    Does anybody know which kit is most tolerant of RIPA buffer while maintaining high sensitivity? The microBCA doesn't have an LOD low enough for my application so I'm looking at the CBQBA.

    Also any lysis buffer alternatives would be welcome though they need to work well on membrane bound proteins.

    Thank you,


    Note: RIPA buffer contents - 25mM Tris•HCl pH 7.6, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS

    Tom Masi · The University of Tennessee Medical Center at Knoxville

    The BioRad DC (detergent compatible) Protein Assay kit works well with a number of different detergents. Although I am not sure how sensitive it is or what sort of sensitivity you are looking for. You can download the manual from their website but it is down until Monday.

    Good Luck!

  • Sudev Naduvath added an answer in Documentation:
    Do you know University Professors of Statistics without any published article as first author in his/her speciality?

    I think it should be documented such cases and the true reason of it.

    Sudev Naduvath · Vidya Academy of Science & Technology

    Dear Sir,

    I have seen many authors in my university who mention the list of authors in their article in alphabetical order of their Last name.  I know a couple of brilliant professors here who have a large number of single author papers and their names have never come as the first author in multiple author papers.  Is it necessary to give undue importance to the order of authors in a research publication?

  • Arianit Reka asked a question:
    What is the difference between tripoli and tripolite?

    What is the difference between tripoli and tripolite?

  • John C Edwards added an answer in 1H-NMR:
    Facing an overlapping problem between the water peak and metabolite peaks in 1H NMR analysis - can anyone help?
    Using a Bruker Avance III HD 500 MHz to make the water peak narrower in order to avoid the overlapping. Do you know how to do it, or if this is the best solution?
    PS: using a room temperature probe.
    John C Edwards · Independent Researcher

     I have had good success with using temperature to shift the water peak downfield. At 80 or 90C the wat er peak will have shifted to 4.3 ppm and should be out of the way. Adding DCl will often shift peaks around in a way that might work out at separating overlapped peaks. I would avoid suppression if the peaks are so close as it will heavily effect the samples signal inensity. Pureshift always sounds good bu may not get y ou any extra separation and there is a HUGE sensitivity loss.

  • Hongwei Liu added an answer in Titanium Alloys:
    How do I decide the invariant line for the beta to alpha transformation in titanium alloys?

    In some paper discussing the morphology and crystallography of alpha variants in titanium alloys, someone give the indices of invariant line like <335>, others give <447>, which seems quite complex. Thus, in titanium alloys which one is the real invariant line?

    Hongwei Liu · University of Sydney

    Dear CHuanyun,

    This is a kind of common confusion to the crystallography of phase transformations. Both of the invariant line [335] and [447] are rational at its discrepancy-angle acceptance criterion for the real invariant line.

    There are two ways to get this index of invariant line as precise as possible.

    The first one is to measure and test the invariant line of alpha-Ti precipitate by putting the specimen at the edge-on orientation of the habit plane. TEM measurement needs to be done very carefully and great patience by double edge-on orientation tilting method if you stil do not know the habit plane. Please refer to Prof Chengping Luo’s paper about trace analysis of double edge-on tilting for precipitation in alloys. At this specially chosen orientation, you can find the longest projection length of the growth direction (invariant line) of alpha-Ti precipitate. By overlapping TEM bright field image and the corresponding electron diffraction pattern, you can easily measure its orientation and get an index as close as the real invariant line orientation. Both [335] and [447] are obtained by TEM measurement at the first step. (you can find the discrepancy angle between [335] and [447 is very small (1.37 deg only).

    The second way to get this index of invariant line is to apply crystallography of diffusional phase transformations. There are many different geometric models or theories to do the pure mathematic calculation of crystallography of diffusional phase transformations. It starts from lattice corresponding followed by accounting phase transformation strain distribution. To now, i) O-lattice theory, ii) 3D invariant line model, iii) Edge-to-Edge matching model, (iv) topological theory for phase transformations and (v) IDE model are all available for you to carry out the calculation. The predicted index of a precipitate invariant line, habit plane, interfacial dislocation structure and mutual orientation relationship are all very close to TEM experimental measurements as long as TEM result is reliable.

    At the end, if you are looking for a real invariant line, both [335] and [447] are not real. They are just approximate directions and very close to the real invariant line. The real invariant line is irrational indexed which is not simply to apply and usually replaced with an integer index expression (it must be very close to the real invariant line).

    Both of the TEM experimental technique and crystallographic calculation are both difficult to beginners and are at the top level of research field of transmission electron microscopy for phase transformations. There is no close answer up to now and still open to explore.

    Hope this can make you clear.

    Kind regards


  • Sharad S Malavade added an answer in Statistical Analysis:
    How to test multicollinearity in logistic regression?
    I want to check multicollinearity in a logistic regression model, with all independent variables expressed as dichotomous.
    Given that I can not use VIF, is the correlation matrix the only possible procedure? If so, what is the threshold for a correlation to be unacceptable? 0.60? 0.70?...
    Sharad S Malavade · Brandon Regional Hospital, Brandon, FL

    I am also testing for multicollinearity using logistic regression. I have all outcomes and predictors as categorical variables. I am using a method described by Paul Allison in his book Logistic regression Using the SAS System. He also gives the SAS code that you can adapt for your use. Adrian mentioned in his post, this method applies weights. The interpretation is then exactly like in linear regression.

  • Mahmoud Zouaoui added an answer in Validation:
    Organisation Values and its impact on Business results?

    I am doing research on the construct of tangible and intangible business impact achieved by rigorous implementation of Organisational Values in letter & spirit in the organisational context. Please suggest published research papers on this subject besides validated construct and questionnaire which can be referred and utilised for validating the above construct.

    Mahmoud Zouaoui · University of Manouba, Tunis, Tunisia

    The following articles could be useful for your study:

  • Timothy A Ebert added an answer in Effluents:
    Can someone help me with the statistics to use in this experiment?

    what statistics can i use in an experiment involving 3 macrophytes and 6 effluents in a remediation experiment,useing the macrophyte to treat  the effluents

    Timothy A Ebert · University of Florida

    I suspect that in each case the outcome will depend on the starting concentration of macrophytes and concentration of effluents. Ono suggested the most basic approach, and this was a good suggestion. That is where I would start.

         If appropriate you could look at the interaction between macrophytes. This is then a mixture design where the total number of macrophytes is a constant and it can consist of 0 to 100% of any one macrophyte.

        It might be appropriate to think of the effluent as a mixture. Different effluent concentrations will have different toxicities, will have different pH, different salt load. They may have different molecular weights, and may be different mixtures of a wide variety of different chemicals.

         You should consider that the efficacy of the macrophytes will be influenced by the availability of suitable nutrients to support growth and reproduction.

         You could consider using population models to look at growth and reproduction rates for the macrophytes. This would tell you if a given macrophyte population will outcompete the other macrophytes in a given effluent.

         It depends a bit on what you are measuring. Are you measuring effluent degradation rates over time as a function of starting macrophyte level/mixture?

  • Sarah Shahmoradian added an answer in Galectin 3:
    What parameters can I study on CSF samples?

    I am collecting CSF samples from neurodegenerative patients.

    I am currently doing galectin-3 and MMP-9 assay. Please suggest other assays as I want to utilize every drop of the precious |CSF samples.

  • Tom Masi added an answer in Genetic Insertion:
    How can I get recombination instead of Gene insertion instead of recombination with pKD46K?

    My lab is trying to knock-out some genes in DH5a using pKD46K.  This plasmid contains a Lambda Red Recombinase system under the control an arabinose inducible promoter.  We make competent cells with pKD46K cultured in LB + L-arabinose, electroporated gene fragment of "5' homology arm - gentamicin resistance gene - 3' homology arm" (HA5-Gm-HA3), and screened for gentamicin resistant clones.  We have repeated this process several times and found dozens of gentamicin resistant clones.  To our surprise, all these clones have the HA5-Gm-HA3 fragment inserted somewhere in the DH5a genome, and none of them showed intended homology-based replacement.  Why?  What can we do to make the homology recombination work? 

    Tom Masi · The University of Tennessee Medical Center at Knoxville

    How many bases comprise each of your homology arms? I have always used 50 bases of homology on each end of my DNA fragment I want to recombine. The overwhelming majority of the resulting transformants contained my DNA fragment in the correct location.

    You might want to try using pSIM6 which is the Lambda Red Recombinase system under the control of a heat inducible promoter (42 degrees C). I initially tried using pKD46 and didn't have much luck with it. Then I switched to pSIM6 and it worked very well.

    Good Luck!