Q&A

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  • How does acculturation impact multigenerational families in therapeutic treatment?

    My interest is in Spanish speaking families from Central America, South America and the Caribbean. 

    Lisa Aronson Fontes · University of Massachusetts Amherst

    Dear Marian,

    YOu ask a complex, multi-faceted question that defies a simple answer. Intergenerational differences are apt to pop up concerning many topics including:

    1. Preferred language
    2. Gender roles
    3. Ideas about how to discipline children
    4. Relationship to social institutions such as schools and healthcare providers
    5. Ideas about health and eating
    6. Ideas about marriage and children
    7. Ideas about sexuality in general and same sex relationships
    8. Celebreating holidays
    9. Proper families roles (balance of power)

    And so much more!

    I suggest you take a look at Celia Falicov's book, Latino Families in THerapy, which has recently come out in a new edition.

    Best wishes with your work,

    Lisa 

  • Edward Koellhoffer asked a question in Pipetting:
    Does anyone run 10uL qPCR reactions?

    I use Bio-Rad's SYBR Green Master Mix for my qPCR work which, according to the manufacturer's instructions calls for a 50uL reaction volume. I use 20uL on a regular basis without any problems. As I am running a lot of qPCR in the future and looking for ways to save some money, I was wondering if it's realistically possible to use a 10uL reaction volume and achieve the same results in terms of well-to-well variability. My main concern is the inevitable increase in pipet ting error as the reaction volume decreases. 

    So, does anyone use 10uL qPCR reaction volumes? Have you ever had any issues?

  • Wen Shiang Lee added an answer in Fermentor:
    Does anyone have experience with Pichia pastoris growth in a fermentor using the AOX1 promoter?

    I'm trying to run fermentation of Pichia pastoris using the AOX1 promoter but so far I didn't get any activity (it's positive clone from shake growth).

    Can somebody tell me how much methanol you added per day? I found one article saying 15 ml/L/hr, which would mean 3.6 l per day for my 10 liter batch. Other articles mostly add it depending on the dissolved oxygen or methanol in the medium, but we do not have way to determine the methanol content, and unfortunately our O2 probe seems to be nonresponsive.

    Right now I'm adding 50mL of 100% Methanol (0.5% of volume) per day.

    Also, did you add more glycerol after it was finished to get the yeast grow even more?

    Wen Shiang Lee · National Taiwan University

    Did you mean 50ml/L-day? could you tell us yeast OD when you start add the methanol?
    I think methanol too less

  • Luke Searcy added an answer in Transgenic Mice:
    Has anyone ever conducted electrophysiological test like LTP test on 3xTg mice?

    According to JAX, it is not recommended to use male 3xTg mice as tested subjects because of the report that male transgenic mice may not exhibit the phenotypic traits originally described. But for eletrophysiological test, male mice are usually preferred due to little estrogen effect. So is it a good method to test their LTP? Thanks a lot!

    Luke Searcy · The University of Edinburgh

    We also ran several electrophysiological experiments in the 3XTg mice, including both extracellular and intracellular recordings at the University of Kentucky in the labS of Olivier Thibault and Nada Porter . 

    See: Searcy et al. J Alzheimers Dis. 2012;30(4):943-61. doi: 10.3233/JAD-2012-111661.

  • Does free access to knowledge and/or pseudo knowledge (googwik science) have damaging effects on society and science?
    A "googwik scientist" is a special case of an amateur of science who, as a source, uses almost exclusively Google and Wikipedia.

    Since choosing a reference from Google, a user is hers/his own judge, and since Wikipedia is unreliable, a product of a consensus of "all" and not only of the experts, such research promotes multiplication of "referenced" mistakes. Sometimes even the references support nonsense. Scientists also use Google and Wikipedia, but since they already know well the background of the studied problem, they read more critically the variety of sources available on Internet and can in fact profit from them. People who are not experts, on the contrary, are bound to accept unscientific or unfounded information as scientific and be victims of the illusion that they are using reliable sources, and falsely believe to have acquired knowledge.

    Is "googwik science" good as a means to increase general knowledge of all and contribute to the advancement of science in general, since it offers, as knowledge, both - correct information, that may be falsely interpreted, or even false information?

    Does science profit from "googwik science" or does "googwik science" in fact damages science by introducing science for all - which in reality permits false interpretation and thereby neither helps to increase knowledge nor to promote science?
    Clifford Miller · Clifford Miller

    Oh, I should have added a link to Googlwik University's Treatise on SBS.  Here it is - complete crap:

    Note for example "Commonly, there are no externally visible signs of the condition".  But of course the infant has been shaken to death with no external signs - not even hand grip marks left on the dead body nor bruising nor even signs of old injury nor any sign of abuse. 

  • Is it possible to measure the conductivity of polymer fibres?

    We want to measure the conductivity of polymeric fibres of 10 micron diameter.

    What is the best way to obtain quantitative and accurate data? Is there any standard setup to do it? 

    Thanks a lot.

    S.M. Korobeynikov · Novosibirsk State Technical University

    The problem of measurement will be in separation of surface conductivity and bulk conductivity. For polymer dielectrics the one is compared with the other. Difference between them could be obtained, E.G., by using of fibers with several diameters. Similar problem was solved in the paper  (J. Phys. D: Appl. Phys. 38 (2005) 915–921 "Surface conductivity at the interface between ceramics and transformer oil" S M Korobeynikov, A V Melekhov, Yu G Soloveitchik,M E Royak, D P Agoris and E Pyrgioti)

  • Mafatlal M. Kher added an answer in Anther culture:
    Can anyone explain why I am getting hairy root development in calli instead of shoots formation?

    Calli generated through anther culture in Brassica Oleracea is showing organogenesis but instead of shoot formation it is going for hairy root. The physical conditions in the lab are 25(+/-2) degree Celsius,12h photoperiod, relative humidity 55(+/-5), hormones: 2-ip and NAA (ratio was 10:1 but changed to 50:1 last week). Are there any suggestions from experts out there? Please, Thank you.

    Mafatlal M. Kher · Sardar Patel University

    Based on your figure it is look like somatic embryo. 

    Just go through following literature

    (Carloni et al., 2014; Cha-um et al., 2009; Escobar-Guzmán et al., 2008; Germanà, 2010; Górecka et al., 2009; Irikova et al., 2011; Jia et al., 2014; Kou et al., 2012; Kozak et al., 2011; Li et al., 2013; Perera et al., 2008; Prado et al., 2010; Redha and Suleman, 2010; Silva, 2009; Winarto et al., 2011, 2010a, 2010b; Zamani et al., 2000)
    Carloni, E., Ribotta, A., López Colomba, E., Griffa, S., Quiroga, M., Tommasino, E., Grunberg, K., 2014. Somatic embryogenesis from in vitro anther culture of apomictic buffel grass genotypes and analysis of regenerated plants using flow cytometry. Plant Cell, Tissue Organ Cult. 117, 311–322. doi:10.1007/s11240-014-0441-4
    Cha-um, S., Srianan, B., Pichakum, A., Kirdmanee, C., 2009. An efficient procedure for embryogenic callus induction and double haploid plant regeneration through anther culture of Thai aromatic rice (Oryza sativa L. subsp. indica). In Vitro. Cell. Dev. Biol. - Plant 45, 171–179. doi:10.1007/s11627-009-9203-0
    Escobar-Guzmán, R.E., Hernández-Godínez, F., Martínez de la Vega, O., Ochoa-Alejo, N., 2008. In vitro embryo formation and plant regeneration from anther culture of different cultivars of Mexican husk tomato (Physalis ixocarpa Brot.). Plant Cell. Tissue Organ Cult. 96, 181–189. doi:10.1007/s11240-008-9474-x
    Germanà, M.A., 2010. Anther culture for haploid and doubled haploid production. Plant Cell, Tissue Organ Cult. 104, 283–300. doi:10.1007/s11240-010-9852-z
    Górecka, K., Krzyżanowska, D., Kiszczak, W., Kowalska, U., 2009. Plant regeneration from carrot (Daucus carota L.) anther culture derived embryos. Acta Physiol. Plant. 31, 1139–1145. doi:10.1007/s11738-009-0332-1
    Irikova, T., Grozeva, S., Rodeva, V., 2011. Anther culture in pepper (Capsicum annuum L.) in vitro. Acta Physiol. Plant. 33, 1559–1570. doi:10.1007/s11738-011-0736-6
    Jia, Y., Zhang, Q.-X., Pan, H.-T., Wang, S.-Q., Liu, Q.-L., Sun, L.-X., 2014. Callus induction and haploid plant regeneration from baby primrose (Primula forbesii Franch.) anther culture. Sci. Hortic. 176, 273–281. doi:10.1016/j.scienta.2014.07.018
    Kou, Y., Ma, G., Teixeira da Silva, J.A., Liu, N., 2012. Callus induction and shoot organogenesis from anther cultures of Curcuma attenuata Wall. Plant Cell. Tissue Organ Cult. 112, 1–7. doi:10.1007/s11240-012-0205-y
    Kozak, K., Galek, R., Waheed, M.T., Sawicka-Sienkiewicz, E., 2011. Anther culture of Lupinus angustifolius: callus formation and the development of multicellular and embryo-like structures. Plant Growth Regul. 66, 145–153. doi:10.1007/s10725-011-9638-2
    Li, Y., Li, H., Chen, Z., Ji, L.-X., Ye, M.-X., Wang, J., Wang, L., An, X.-M., 2013. Haploid plants from anther cultures of poplar (Populus × beijingensis). Plant Cell, Tissue Organ Cult. 114, 39–48. doi:10.1007/s11240-013-0303-5
    Perera, P.I.P., Perera, L., Hocher, V., Verdeil, J.-L., Yakandawala, D.M.D., Weerakoon, L.K., 2008. Use of SSR markers to determine the anther-derived homozygous lines in coconut. Plant Cell Rep. 27, 1697–703. doi:10.1007/s00299-008-0592-z
    Prado, M.J., Grueiro, M.P., González, M.V., Testillano, P.S., Domínguez, C., López, M., Rey, M., 2010. Efficient plant regeneration through somatic embryogenesis from anthers and ovaries of six autochthonous grapevine cultivars from Galicia (Spain). Sci. Hortic. 125, 342–352. doi:10.1016/j.scienta.2010.04.019
    Redha, A., Suleman, P., 2010. Effects of exogenous application of polyamines on wheat anther cultures. Plant Cell. Tissue Organ Cult. 105, 345–353. doi:10.1007/s11240-010-9873-7
    Silva, T.D., 2009. Indica rice anther culture: can the impasse be surpassed? Plant Cell. Tissue Organ Cult. 100, 1–11. doi:10.1007/s11240-009-9616-9
    Winarto, B., Mattjik, N.A., Teixeira da Silva, J.A., Purwito, A., Marwoto, B., 2010a. Ploidy screening of anthurium (Anthurium andreanum Linden ex André) regenerants derived from anther culture. Sci. Hortic. 127, 86–90. doi:10.1016/j.scienta.2010.09.004
    Winarto, B., Rachmawati, F., Pramanik, D., Teixeira da Silva, J.A., 2010b. Morphological and cytological diversity of regenerants derived from half-anther cultures of anthurium. Plant Cell. Tissue Organ Cult. 105, 363–374. doi:10.1007/s11240-010-9876-4
    Winarto, B., Rachmawati, F., Teixeira da Silva, J.A., 2011. New basal media for half-anther culture of Anthurium andreanum Linden ex André cv. Tropical. Plant Growth Regul. 65, 513–529. doi:10.1007/s10725-011-9622-x
    Zamani, I., Kovacs, G., Gouli-Vavdinoudi, E., Roupakias, D.G., Barnabas, B., 2000. Regeneration of fertile doubled haploid plants from colchicine-supplemented media in wheat anther culture. Plant Breed. 119, 461–465. doi:10.1046/j.1439-0523.2000.00538.x

  • Eleni Tracada added an answer in Spatial Planning:
    Does anyone see differences between "strategic planning" and the "strategy in spatial planning"?

    The relation between these two kinds of plans are very important but in a lot of cases we don't have any strategic plan previously to a Spatial Plan. So we need to put a strong strategic component in the spatial plan (normally a Municipal Master Plan). But I think that it is very different.

    Eleni Tracada · University of Derby

    I agree with both Elisabeth and Valentina above.  However most of the times a strategic vision is imposed by top-down rather than bottom-up processes unfortunately. The European Charter defines spatial planning by emphasising 'expression to the policies of society'.  Thus, society and especially local communities' consultation should be the first and most important part of these processes, if we wish to get an inclusive strategic vision. 

  • Pawel Mroczkowski added an answer in Lymph Nodes:
    How can I evaluate N score in pTNM when an insufficient number of lymph nodes are removed? For example -only 4 l.n. in rectetomy etc?

    If there are only 4-5 lymph nodes and all of them are negative, how do I consider N?

    Pawel Mroczkowski · Otto-von-Guericke-Universität Magdeburg

    There are two aspects - the formal one - Glenn described it extinsevely, there is nothing to add. Clinical more interesting is the different aspect - what ist the reason of this low amount. The pathologists blame surgeons and the surgeons blame pathologists. The way to solve it is usage of a standardised pathological work-up of the specimen (the standards of the Royal College of Pathologists are a very good option), it the numbers are still low then to talk with the surgeons.

  • Krishnashish Bose added an answer in Sorting:
    Is there a way to sort a list of {x,y} coordinates based on both x & y values?

    p={{17,19},{18,18},{19,17},{20,15},{20,16},{21,14},{22,13},{23,13},{24,12},{25,12},{26,12},{27,12},{28,12},{29,12},{30,12},{31,12},{31,19},{32,12},{32,19},{33,12},{33,19},{34,13},{34,19},{35,14},{35,18},{36,15},{36,16},{36,17}}

    This list has been sorted based on x-values. I can sort them also based on y-values. But I cannot sort them considering both x & y values simultaneously.

    I will give an example
    {19,17},{20,15},{20,16},{20,17}
    they should be arranged like:
    {19,17},{20,17},{20,16},{20,15}
    u see that neigbouring points allow only one unit of change in either x,y or both

    Krishnashish Bose · Nanyang Technological University

    You are right. They are sorted along a path through all the points in which the individual steps are either (1,0) or (0,1) or (1,1). All the points can be connected by a unit difference as they belong a connected contour. Please check the attached file.

  • Does anybody know how to screen all the chemical compounds found in the herbal plant?

    I need a proper method how to prepare the sample before I run them through GC-MS analysis.

    Hi Noor

    Natural products from plants serve as rich resources for drug development with almost 100 plant-derived compounds in clinical trials in 2007. Plant derived natural products have had a profound and lasting impact on human health and include compounds successfully used for decades such as digitalis, vincristine, Taxol and morphine isolated from foxglove, periwinkle, yew, and opium poppy, respectively. The enormous structural diversity and biological activities of plant-derived compounds suggest that additional, medicinally relevant compounds remain to be discovered in plants.For more please read at the following links.

    Best regards

  • Media for UCB1 pistachio micropropagation?

    Hi, i started working with UCB1 pistachio micro propagation. I made some media according to Parfitt, D.E. and A.A. Almehdi. 1994.(i've attached the link to the paper).  The media is composed of DKW supplemented with 30g sugar, Garmborg's vitamins, 85.5mM Zn, 156mM B and 9.9mM KNO3. Although the plants managed to grow, they don't callus properly. The stem above the media become swollen and the stem below the media just turn black. Over time, the stem just become woody/hard and turn reddish. The leafs also looks vitrified to me. I've tried to culture the plants on MS and 1/2 MS with no success. Can anyone suggest any papers or protocols i can follow to micro propagate them? Thanks!

    Cecília Antão da Silva · University of Lisbon

    Hello, Iu Wang

    From your description, it seems rather a physics problem than chemistry. Have you tried to reduce the water in the medium, decrease sugar content, and change to 8h photoperiod? That might reduce vitrification and oxidation of the explants while increasing the number of calli. Good luck!

  • Alessia di capua asked a question in Fluoresceins:
    Fluorescein in Supercritical Assisted Atomization?

    some articles about it? 

  • Can anyone help in "artificial neural network" algorithm?

    I would like to know the basic details of this algorithm. I found various papers but failed in understanding the actual algorithm. 

    Thank you in advance. 

    Evon Abu-taieh · University of Jordan

    See these PPT they might help.

  • Ľubica Krajčíová added an answer in Primer:
    Before adding the restriction sites, my primers have a 9°C Tm difference but after adding them, the Tm values narrowed. How does this affect my PCR?

    The primers that I designed initially had Tm values of 54.26°C (Fwd primer) and 45.53°C (Rev primer) before I added the restriction sites. After I did that, however, I managed to get the Tm adjusted to 68.59°C and 69.54°C for both the forward and reverse primers respectively. This was done using the Primer-Blast tool. I do realize that the difference in the Tm value is quite large, but I'm not sure whether this will severely affect the PCR.

    Would I have to set a lower annealing temperature for a few cycles first before increasing it to the Ta for my complete primers (including the restriction sites)?

    Ľubica Krajčíová · Univerzitná nemocnica Bratislava

    I think that design a new primer will be the best choice. You can find the another second primer, which will has a better Tm. You can create a new primer close to the previous one (only a few nucleotides) it can  help.

  • Peter T Breuer added an answer in Vedic Mathematics:
    Is it possible to implement any reed solomon codec using vedic mathematics?

    How vedic mathematics can solve the problems errer correcting codes. i.e. is there any application in finite field like to solve the 'Key Equation solver' block of RS code.

    Peter T Breuer · Birmingham City University

    Out of a possibly ill-advised mild sense of curiousity,  I have to ask what 'vedic mathematics' may be when it is at home.

    Google thinks it's a set of about 9 mental arithmetic tricks. So I presume `vedic mathematics' would mean just doing some sums mentally, using these particular operations. What would be the point of that? Indeed, what does "solve the problems errer (sic) correcting codes" mean? Do you mean, APPLY an error-correcting code (to correct an error)?

    And what is a "Key Equation solver"? What is a "RS code"? Please remember to define terms of art before the point of first use if you use any. They are jargon.

  • Wai Sun Don added an answer in Finite Volume Method:
    Is it possible to get second order accuracy at the boundaries using finite difference method?

    I read recently that "it is not possible to get second order accuracy at the boundaries using finite difference method, were as same is possible with finite volume method. Which by the way is one of the differences between finite difference and finite volume method." Can anyone give me a better insight into this?

    Thanks!

    Wai Sun Don · Ocean University of China

    You should 

  • Are my CT scans of an Infrarenal thrombosed saccular abdominal aortic aneurysm an Indication of surgery?

      Paitent is a 61year-old man who underwent AVR due to AR recently. CT scan before AVR showed infrarenal saccular abdominal aortic aneurysm which maximum diameter was 40mm. Saccular part of this aneurysm was almost thrombosed. AVR done successfully.

     Patient is asymptomatic and does not have any particular history of systemic infection, auto-immune disease, or connective tissue disease. Pathology of this aneurysm seems to be atherosclerotic.

     Open surgery? Stent? Observation?

    Turki Alfuhaid · King Fahad Medical City

    Follow up with Ultrasound every 6 months is the best approach unless the patient is symptomatic (pain or emboli). Based on size criterion, once it reaches 5 to 5.5 cm then surgery should be considered. Measurements must be done by a technologist with good experience with vascular ultrasound since we are talking about millimeters changes. 

  • Is it possible to remove AMONIL (thickness about 80nm) with a RIE process using CF4 gas? What conditions do you use?

    I'm trying to optimize the fabrication of silicon based supports obtained by soft UV-NIL and reactive ion etching (RIE) using AMONIL as resist.I have some problems (rugosity) when I try to remove AMONIL by RIE using CF4 gas or a mixture of CF4/Ar gas .

  • How soon after major cardiac surgery should the drainage tube be removed?

    By major I mean a cardiopulmonary bypass machine was used?

    Yves Durandy · Centre Chirurgical Marie Lannelongue

    I agree with the others. In the vast majority of cases chest tubes are remove on day 1  the drainage being clear and negligible and the chest X ray demonstrating no retention. We are more reluctant to remove chest tubes early when there is a need for heavy anticoagulation, like for a mitral valve replacement in a patient with chronic atrial fibrillation. We will probably keep the chest tubes until stabilization of oral anticoagulation.

  • Yoram Oron added an answer in Cell Count:
    What is the most precise method for cell counting?

    It seems that the method of cell counting with a TC20 cell counter by Bio-Rad is associated with with high variations. Therefore, I am searching for a better method to improve the accuracy. Can anybody suggest a precise method for cell counting or recommend a paper where the different methods were compared?

    I would like to count neuron-like cells in a collagen-coated 6-well plate.

    Thank you for your help, Alisa

    Yoram Oron · Tel Aviv University

    Dear Jair,

    Counting cells that are highly irregular (e.g. neurons) is difficult either manually or by an automated method. In my opinion the best would be to stain nuclei with DAPI and count either manually with a fluorescent microscope or , after taking a frame, by any reasonable software (e.g. Metamorph) after thresholding and converting to binary image. If your support is blocking UV, there are 96 clusters designed for UV.  If you do not have access to fluorescent microscope, you can count nuclei on a regular H&E staining, but only manually since no software is good enough for that.  If you do not want to kill the cells, you can use Hoecht's stain, which is supravital. You'll stil need fluorescent scope for that.

    Hope that helps

  • Benet Dhas added an answer in Bisulfite Sequencing:
    Can anyone explain how to calculate methylation level from bisulfite sequencing results?

    I have followed all the standard protocols for bisulfite sequencing like cloning, etc. I got the sequence and I can identify whether the CpG is methylated or unmethylated but I don't know how to calculate the percentage of methylation from the sequence electrophoregram. How do I do this?

    Benet Dhas · Jawaharlal Institute of Postgraduate Medical Education & Research

    Thanks Wagner. I had done bisulfite specific PCR, gel eluted the specific product and cloned it. I got more than one clone but I sanger sequenced only one clone. Do I have to sequence all the clones to find out level of methylation?

    I am bit confused. A CpG can be a methylated one or unmethylated one. How can it be a partially methylated. If I am looking into a single CpG, is it possible that I get partial methylation? Kindly clarify the term PARTIAL METHYLATION

  • Sumeet Gupta added an answer in Personality Traits:
    Can I reduce the number of items for further analysis when the model fit of the established scale isn't good enough?

    We are currently doing empirical research on personality and entrepreneurship. We used the established BFI-44 to assess the respondents Big Five personality traits. Before I start now hypotheses testing, I conducted a confirmatory factory analysis (CFA) with the result of a relatively poor goodness of fit (GOF). I could now exclude some of the items in order to increase GOF and then continue my hypotheses testing with the reduced number of items. However, I'm not sure if I may simply exclude those Items as they originally belong to the established scale. So may I or may I not? Does anyone have literature on this question? Thank you in advance!

    Sumeet Gupta · Indian Institute of Management Raipur

    Yes you can. Please check Hair et al.'s Multivariate Data Analysis (Seventh Edition) or LISREL Modeling by Anderson and Garbing. 

  • Emilia Bergen asked a question in Cognitive Training:
    Any experimental research on improvement (or lack thereof) of adolescents’ decision-making skills for long-term goals rather than short-term goals?

    Research on individuals aged approximately 10-17, cognitive training tasks, pre and post testings, generalizability of the training, anything on the subject really.

  • Alessia di capua asked a question in Fluoresceins:
    How much fluorescein should I use to label it to the BSA?

    I would like to know how much sodium fluorescein and BSA should I use in an aqueous solution. 

  • Giulio Mecacci added an answer in Free Will:
    What is the difference between a "cue" and a "cause"?
    I am puzzled by the definition of "cue" as opposed to "cause". I am reviewing the literature about self-generated action (Passingham et al 2010; Nachev 2010; Shuur & Haggard 2011). The authors talk about cues extensively, as conditions that prompt an action. My question is, in what way does a "cue" differ from a "cause"? I keep missing a deep grasp on this.
    Giulio Mecacci · Radboud University Nijmegen

    You say that cues are non-sufficient conditions for an action. I would add they are also non-necessary as the same action can be prompted by different cues. I guess that much depends also on the granularity of the way cues are specified. More investigation needed, thank you for your time!

  • Sergey V. Dorozhkin added an answer in Scaffold:
    Does anyone know a good Hydroxyapatite slurry for filling a mold in order to make a trabecular scaffold?

    I am trying to print the negative of bone trabeculae then fill with hydroxyapatite to create a scaffold to seed cells on but haven't found a good HA slurry to use. 

    Normally “a good HA slurry” appears to be a multi-component suspension. Namely, besides fine HA powder and water, it contains various additives such as binders, surfactants, anti-foaming and anti-coagulation agents, etc. to simplify the production state. Usually, such HA slurries are used by scaffold producing companies and, therefore, both the precise compositions and preparation receipts are known but kept in a secret. Therefore, try to look in publications and/or Internet and, in the case of a failure, you will need to develop it by yourself.

  • Any help with my bacterial transformation problem?

    I transformed the cry2A gene (1.9kb) into the E. coli JAS86 strain using pET32. I got around 32 colonies onto 4 plates of LB medium supplemented with ampicillin (50µg/ml) and kanamycin (60µg/ml). I subcultured the colonies on LB medium with amp and kan antibiotics.

    Next, I plasmid isolated 25 isolates and digested with EcoR1 and BamH1 (single and double digestion). But none of this has yielded me a single plasmid of around 8 kb or two plasmds (5.9kb+1.9kb).

    This E coli strain already contains the plasmid with 7140 bp (being present in upto 5 copies per cell). It seems I am just getting the backbone of the pET vector (around 5-6 kb aftr digestion).

    Can anyone please tell me how can verify my recombinant clone with digestion? Should I repeat the transformation?

    I have uploaded the gel photo after digestion. 1 kb ladder used (200, 500, 1000, 2000, 3000, 4000, 5000, 6000, 8000, 10000bp)

    Cara Pina · Brandeis University

    I'm having a hard time telling if you are having a cloning problem or a transformation problem. 

    It seems like you are getting transformants (cells that take up the pET32 plasmid) but you are having problems cloning your gene of interest into the pET32 backbone. Or do you already have the completed pET32 plasmid + cry2A and when you transform you aren't getting isolates containing cry2A?

    also, what method of transformation are you using? 

  • Nadine A. Gund added an answer in Bioanalyzer:
    Is it possible to perform fragment analysis of SSRs through Agilent Bioanalyzer 2100

    I would use DNA 1000 LabChips. I couldn't find articles suggesting the usage of this technology to analyse variation of alleles in populations.

    Nadine A. Gund · Bayerische Landesanstalt für Landwirtschaft

    Hi, ask the company. They will give you the answer. Cheers, Nadine