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Hi,
I am using a technique in C2C12 muscle cells called DamID to tag DNA in close proximity to the nuclear envelope in undifferentiated myoblasts and differentiated myotubes. However, following sequencing of the tag-enriched material amplified by the final PCR step, 80% of the DNA sequences identified come directly from the mitochondrial genome.  So it looks like the mtDNA is being tagged and because per copy it is more numerous than any genomic sequence, gets preferentially amplified.
I have tried removing the circular and supercoiled mtDNA by;
  1. subtractive hybridization (purifying mtDNA, fragmenting it, biotinylating it, hybridizing it material to my sample and then pulling down the hybrid duplexes with Dynabeads)
  2. Nuclear isolation (hypotonic lysis, dounce homoginisation, centrifugation)
  3. CsCl gradients (using the density difference acquired by linear and supercoiled DNA following addition of ethidium bromide)
However, none of these have worked. Does anyone have any suggestions on easy ways to remove the mtDNA from the genomic DNA? The last thing I am thinking of trying is gel filtration?
Any help would be great!
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I find one paper use Cas9-assisted removal of mtDNA.
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I am currently searching for protocols to do phage isolation (of all phage present) from an environmental sample (water), and CsCl is recommended for ultracentrifugation. I am trying to determine if Percoll may also work for setting up the gradient. If not, why is CsCl better for collecting phage?
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I assume Percoll would work in a gradient with phage but have never used it. Both CsCL or Sucrose gradients can also be used for concentration of phage from a water samples. Alternatives to this are to use filtration based methods using Amicon columns or iron ch flocculation of the phage/viruses, depending what you want to do with the phage at the end of the process. 
These methods have been developed by Matt Sullivans lab and details can be found here https://u.osu.edu/viruslab/protocols/#VirusPurificationandConcentration 
These are based on Marine water, but work with water from other environments too 
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During phage dialysis,its been written somewhere that dialysis process helps to remove CSCL particles from phages,but how its possible as phage size is smaller than cscl , so if cscl moves out from membrane then phage too can move outside?
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Cesium chloride (CsCl) is a salt with molecular weight 168. Phage are very large macromolecular complexes. CsCl is easily removed by dialysis. Phage will not pass through dialysis membrane (typical molecular weight cutoff is 10,000 Da).
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I try to purify virus-like particle that express in recombinant yeast.
By sucrose cushion, then subject to equlibirum density CsCl centrifugation.
After I culture yeast for 100mL, harvest, break cell by beat beater, then clarify the lysate by centrifuge at 11,000 rpm for 10 min. Treat Dnase and Rnase, 37 C, 30 min. Then, 14,000 rpm for 20 min. Finally, use 0.45 um membrane filter.
For ultracentrifuge. I layered the lysate on 40%w/v sucrose in PBS buff. Centrifuge 27,000 rpm, 2 hours.
I confuse with the result (already attach image file below). I see a lot of white pellet in the phase of sucrose (middle), and very little pellette in the bottom of the tube. Then I try to suck all solution out, but after the tube is empty from solution, I can not see the pellette in the bottom any more.
The questions:
1. The pellette in the bottom (very little amount) is my virus particle or not?
2. The bulk pellete in the middle layer is the cell debris or not?
3. Should I treat Dnase and Rnase, It will have a bad things with my expermient or not? Normally, I see on paper, no one treat its.
Any suggestion, please provided
Thank you.
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Hi,
Treatment with DNAse RNase may not be required,
Viruses /VLPs differ in their density based on which you need to decide on the sucrose % of cushion.
If your VLP is denser than the cushion you used the pellet you are observing contains your VLP.
All other proteins cellular fragments whose density is less than the cushion density are observed as layer over the cushion.
Finally, if your cushion is less denser than VLP all your observations are correct  
Hope the information is useful
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Hi everyone. I have cryo EM sample containing DNA-bacteriophage. The bacteriophage is purified in CsCl gradient and concentrated in a centricon concentrator with 100 kDa cutoff. The problem is that I can see high DNA contamination in the background. If a DNAse is added to my sample all bacteriophage particles are gone.  Therefore, I need to purify it some other way. Any ideas? 
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you are right Dominic I misunderstood the question and thought we were dealing with  a mixed dna prep from an em sample and was just trying to get rid of the genomic dna without dnase
Good luck
paul
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Hi,
I have been doing EPSC/IPSC recording from CA3 pyramidal neurons in acute slices using a CsGluconate internal solution (in mM): 
130 Cs-Gluconate, 10 HEPES, 0.6 EGTA, 3 MgCl2, 1 NaCl, 0.4 CsCl, 4/0.3  ATP/GTP, 10 phosphocreatine and 1 QX-314 (recording EPSCs at -70 mV in voltage clamp, IPSCs at 0 mV).
My colleague, who has been doing electrophysiology much longer than I, uses for the same experiment:
140 K-gluconate, 10 HEPES, 1 EGTA, 4 NaCl,  4/0.3  ATP/GTP, 10 phosphocreatine and 1 QX-314 (recording EPSCs at -65 mV in voltage clamp, IPSCs at 0 mV).
When I calculate the reversal potential for Cl-, mine comes to -73 mV and his to -89 mV (we both use the same ACSF with APV).  I can’t get a clear answer from him as to why he uses this solution and concentration of Cl-. 
Can anyone provide insight into this?  If so, I would greatly appreciate it.
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For reliable recording it is important to use K-gluconate. In addition to the problem of juction potential,  Cs blocks K channels and causes decrease of resting potential. So, parts of the cell, which you can't properly clamp (this is usual problem at whole-cell recording from neurons) will be artificially depolarised. 
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Is CSCL Model or Framework exists? or to learn CSCL Model build up from e-learning model?
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On the educational perspective, we should consider the collaborative learning theory.
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The idea is to link certain structural indicators of activity with the depth of the messages that relate participants in virtual learning environments. We are working on a project FONDECYT gives us insight into these relationships. Thank you to contact us in case of interest or related work.
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Hello Sthepani
indeed the nature of knowledge is important, but we have not worked that particular aspect but rather the type of educational influence exerted by the teacher. Our work mainly deals with the mechanisms of educational influence and distribution of these mechanisms in virtual environments. The courses analyzed are varied and are now analyzing a particular course programming and teaching a course in social psychology.
 
Thank you for
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Interested to ask for opinions on jigsaw in CSCL
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Dear Asmara,
following research paper aplies and examines the transferability of the Jigsaw collaborative learning technique to a Virtual Learning Environment  in Second Life platform. 
1. Fostering collaborative learning in Second Life: Metaphors and affordances
2. Addtional papers about the Jigsaw technique:
1. Aronson, E., & Bridgeman, D. (1979). Jigsaw groups and the desegregated classroom: in pursuit of common goals. Personality and Social Psychology Bulletin, 5, 438e446.
2. Aronson, E., & Patnoe, S. (1997). The Jigsaw classroom: Building cooperation in the classroom (2nd ed.). Longman, ISBN 978-0673993830.
3. Barkley, E., Cross, P., & Howell, C. (2004). Collaborative learning techniques: A handbook for college faculty. Jossey-Bass. 10:0787955183.
Good luck with your research.
Lia
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I'd like to use interactional data: problems solved, answers given, time taken, concepts involved, certificates received as well as interaction among its users (forum messages exchanged) to run some data analysis.
Does anyone where I can find such dataset?
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Hello Bruno,
Check this link it may help you : http://datastage.stanford.edu/
Best of luck
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1. What are the basics/standards of a CSCL framework?
2. In what area can an agent contribute in a CSCL framework?
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In my opinion, you can do both. But jumping into existing framework will be less helpful as you did not fully understand the components that should be in the TF. Maybe you can decide on either one theory (SConstruct or SocioCultural) then after deciding, explore more about the theory and what they say about CL.
As much as I would like to help, but I am currently working in Amsterdam until Nov 2015. You can come to Amsterdam if you have time ;) 
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With the current existing of CSCL system, many research paper proposing the new framework used in education setting. What are the latest or updated of CSCL framwework can be used as guidance?
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One of the most advanced examples of CSCL as facilitator for "knowledge construction" is Knowledge Forum (or its predecessor  CSILE) developed by Bereiter and Scardamalia. A brief introduction, you will find in: Scardamalia, M. (2004). CSILE/Knowledge forum®. Education and technology: An encyclopedia, 183-192.
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There are some isolated phages for foodborne pathogens. Before characterizing these phages, do they need to be purified? Is there any alternative for CsCl or is it the method of choice?
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Thanks Ariane.
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Some suggest measuring refractive index, some suggest collecting all fractions and selecting virus-rich fraction by qPCR.
I'm wondering why after purification I do not see the formation of a distinct band, like adenovirus, after the same procedure?. 
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We use a step gradient with 5 mls of 1.5 g/mL on the bottom, 12 mls of 1.3 g/mL in the middle, and then layer our vector containing sample (15-20 mls) on top.  After the overnight spin we pull the vector bands (can be hard to separate the empty and full bands), add them to a new tube, fill the tube with 1.4 g/mL CsCl and then do an isopycnic spin overnight.  This should give two nice bands.  Note that we do this routinely for AAV5, but this will also be the same for AAV8. 
If your titers are low, then it will be difficult to see a band. We use a bright light under the tube to help us visualize the band.  You will need to have a titer of at least 1e13 vg to see a reasonable band, and the more the better. 
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Has anyone compared the effects of different plasmid purification methods on the mammalian cell transfection efficiency? I am trying to make MDA-MB-231 cell lines that stably express my proteins. After G418 selection and expansion, the western blot showed me big variations in the expression level in different constructs (these constructs are only different by one amino acid). It turned out the good expression came from the CsCl purified plasmid and the bad expression came from the Qiagen Mega Kit. Both DNAs looks very pure from the OD reading and gel running. Can any one tell me, from experience or theoretically, whether these two purification methods would make the difference? Your answer will help me decide whether I need to do CsCl plasmid purification all the time in the future.
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My recommendation would be to use endotoxin free purification kit. Any suppliers will be OK but it is much better for transfection with endotoxin free kit. The importnat criterai also is the amount of supercoiled form in the preparation. I hope this can help