Questions related to CSCL
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;
- subtractive hybridization (purifying mtDNA, fragmenting it, biotinylating it, hybridizing it material to my sample and then pulling down the hybrid duplexes with Dynabeads)
- Nuclear isolation (hypotonic lysis, dounce homoginisation, centrifugation)
- 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!
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?
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?
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.
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
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?
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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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?
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?
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?.
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.