The study of the molecular processes of the nucleus and cell function.

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107 views

Finding a easy and cheap method for dyeing dNTP

I want to measure OD to know the concentration of dNTP. Any idea for dyeing dNTP at cheapest price and easiest way ?
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1answer
104 views

Preparation of normal DNA polymerase

i don't need Taq polymerase. Just need normal DNA polymerase. So the main idea is preparation polymerase directly from Lactobacillus without using cloned vector. My idea is electrophoresis using ...
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0answers
131 views

Can Pfx polymerase add only one 3' A overhang?

I am trying to clone a PCR product that was amplified using Pfx polymerase into pGemT vector. I had to A-tail the PCR product using Taq polymerase since Pfx only generates blunt end products. My ...
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2answers
77 views

How does the DNA know which chain has error in repairing?

As we know, the DNA has more stability than RNA, if one chain has broken or accidentally distorted, it can be repaired by the other one. Suppose there is a segment AGTC, its peer is GACT. Now its ...
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38 views

Protocol for immobilize oligo to solid

I'm learning about microarrays and understand that an oligonucleotide can be immobilized to a solid like a CD acrylic plastic by modifying the oligo. Does anyone have a good protocol for modifying a ...
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1answer
48 views

Carotenoid biosynthesis in yeast

Does Budding Yeast Sacchromyces cerevisiae produces significant amount of carotenoids? Have anybody estimated the ratio of flux going in branches 1. Cholesterol synthesis(via squalene) 2. Coenzyme Q6 ...
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1answer
54 views

Some question about materials for PCR

I'm planning to order oligo, polymerase, nucleotide, buffer for my first diy PCR experiment (now I have nothing), so I have some questions. How and how long I can storage them? I have only a fridge ...
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2answers
187 views

Can PCR tubes be made of different materials than plastic?

Does it matter if I replace the PCR tube (usually made of plastic) with another material like aluminum, glass, or something similar?
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40 views

Are stable proteins tagged by ubiquitin?

What would happen if an extremely stable protein was created because of some error? Would that protein be tagged by ubiquitin?
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3answers
117 views

Pump to separate protein/DNA instead of electrophoresis?

I know that DNA molecules or proteins can be separated in electrophoresis because the electrical charge is used to pull the DNA through the gel. So instead of using electrical, can I using physical ...
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3answers
126 views

Finding a template/oligo combination for my first PCR experiment

I'm an information technology engineer. I love biology so I research biological topics and have an interest in PCR. That's why I have decided to create a PCR machine. Everything is done now and I ...
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1answer
691 views

What is the exact principle of capillary isotachophoresis?

I know it's a type of capillary electrophoresis, but I don't get how does the separation happen exactly.
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2answers
5k views

Agarose vs agar? Why do DNA gels use agarose only and how do you obtain agarose from agar?

Agar is a relatively cheap substance from red algae. And it contains a saccharide agarose as well as a small amount of pectin. Agar is used for culture plates as is, but for DNA gels a grade of ...
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1answer
651 views

Why is allolactose the LacI inducer?

For what reason(s) is allolactose, instead of lactose, the "natural" inducer of lac operon repressor?
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3answers
419 views

Why do neurones use chemical signalling at synaptic junctions?

Problem. When a neurone fires, it sends an electrical signal that jumps down the axon via the nodes of Ranvier very rapidly. At a synaptic junction, chemical brownian diffusion signalling with ...
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1answer
65 views

How can I save bacillus strains on filter paper without an -80 degree freezer?

I want to save my bacillus strains but I don't have access to a -80 degree freezer. What are possible alternatives?
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1answer
457 views

How do viroids propagate to other hosts?

As viroids are simply non-encapsidated non-coding RNA molecules, it is difficult to imagine a mechanism for their continual infection, other than horizontal transfer which doesn't seem to be the case. ...
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0answers
631 views

Low RNA yield, low 260/230 ratio

I extracted total RNA from animal tissue using the Qiagen RNeasy kit, however my RNA yield was extremely low and the 260/230 ratio was around 0.3. This is the protocol I followed: The animal ...
3
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2answers
518 views

Just how light-sensitive is ethidium bromide?

One lab I was in was paranoid about keeping it in a foil-wrapped conical tube; my current lab leaves it out on the bench (and it works fine for staining gels). It's the same company/concentration in ...
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1answer
54 views

Intracellular lipid transport

I know that lipids are carried around the body in the blood either as micelles or by lipid-binding proteins which allow them to be solved. Lipids can't always be integrated in a membrane though, the ...
2
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1answer
461 views

Optogenetics - How do microbial opsins work?

I'm just introduced to the optogenetics method and am having some trouble grasping the genetics (of the optogenteics) part of things. So we have Retinal and Opsin that form Rhodopsin molecule that ...
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2answers
14k views

What is the function of the RNA primer in DNA replication?

During DNA replication, RNA primase puts an RNA primer in the lagging strand. What is the function of this RNA primer? Why can't the enzymes put DNA fragments directly?
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1answer
40 views

In vitro enzyme production

I need to express a protein in vitro but I don't know where to start. I will likely do a T7 transcription protocol but for translation I am not sure what to do. Are there any good kits?
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0answers
155 views

What mammalian cell lines are easiest to grow and transfect?

I'm working with HEK293 cells, which are very easy to transfect. Does anyone have other suggestions for cell lines with very high transfection efficiency? Ideally the chromosomes would also be diploid ...
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3answers
871 views

Knockdown of long noncoding RNA (lncRNA) - how is it done?

I don't work at the wet lab and don't know all the details about the knockdown techniques. My question is: How lncRNA knockdown is done? For example - you have lncRNA that is functional in the ...
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1answer
678 views

Why is Sanger sequencing inferior for detecting SNPs in cancer cells?

I am familiar with Sanger sequencing, but at the level of an undergraduate. A lecturer of mine tried to describe Sanger sequencing as losing the sequence information in noise when used to detect ...
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1answer
326 views

What is solid-phase bridge amplification?

During Illumina sequencing there is a step called bridge amplification by which DNA is amplified by isothermal enzymes. What is this stage, and how does it work?
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1answer
78 views

How can I clean phenol extracted RNA?

I extracted RNA from skin and quantified it in an Nanodrop machine. The 260/230 ratio was very low and two peaks appeared in the absorbance curve, but the RNA is ressuspended in RNAse free water. How ...
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1answer
33 views

Amount of reverse transcriptase in µg or mM for qRT-PCR

I am trying to calculate a titration amount for a molecule which I would like to use in my PCR samples. Different molecules have different densities so I would like to calculate the appropriate ...
3
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1answer
109 views

High protein turnover rate and protease inhibitors?

I work with mice, and I want to see what happens to some specific proteins in the mouse brain after IL-1b injection (intracerebroventricular). I have a problem: when I measure the mRNA and protein ...
9
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2answers
4k views

Why is PEG important for efficient yeast transformation?

One way to do an yeast transformation is by using lithium acetate, a single-stranded carrier DNA, and PEG (1). I was wondering why is the polyethylene glycol important for the efficient ...
3
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1answer
131 views

What molecular processes are involved in pseudopodial extension?

I am curious as to the processes and mechanisms involved in the extension of pseudopodia in amoeba. How does the cell know and control the direction and extent of pseudopodia formation at a molecular ...
2
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1answer
408 views

Manipulation of gene expression using VP16 fusion and engrailed fusion to a transcription factor?

Today, a presenter briefly mentioned that gene expression in sea urchins during development might be manipulated using VP16 and engrailed fusions. On a slide, it said that expression might be ...
5
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1answer
1k views

How does formaldehyde cause protein-DNA crosslinking?

How does formaldehyde cause protein-DNA crosslinking? I would guess it's because the strongly polar water molecule interacts strongly with polar residues on a protein-DNA complex, and adding a less ...
3
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1answer
95 views

205 nm UV-Vis readings

Typically we determine the concentration of proteins using a 280 nm reading. However, it is reasonable to use 205 nm. I was curious about the effectiveness of this method.
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1answer
232 views

How Antioxidants affects Human Metabolism?

I'd like to know how antioxidants affects the human metabolism and which ones are essential for metabolism processes.
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1answer
232 views

Why does the hydrolysis of ATP increasing entropy increase the Phosphoryl-­‐transfer Potential?

I am currently taking a biology class. I do not understand this concept. I understand that the electrostatic repulsion of the negative charges, resonance stabilization and hydration stabilization all ...
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0answers
45 views

Experience with Detox Cloning

I just saw this paper. A Highly Efficient Molecular Cloning Platform that Utilises a Small Bacterial Toxin Gene I was curious if anyone had experience with this cloning strategy using the IbsC ...
2
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3answers
960 views

Improving Gel Extraction yields

How can I improve my Gel Extraction yields. We use the standard protocol from Qiagen, gel extraction, dissolve in QG buffer at 42C and purify via anion exchange columns. However, with 500 ng we ...
4
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1answer
173 views

What is a simple protocol for staining cells in suspension?

I am an engineering student studying how electric fields affect cells, specifically the phenomena of electroporation in living cells. I know that electroporation is widely used for introducing genes ...
3
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0answers
77 views

How does LCR compare to Assembly PCR

The question pretty much explains itself. How do the two methods compare? I've always used Assembly PCR but the method is prone to mistakes and I'm curious how it compares to Ligase Chain Reaction ...
9
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1answer
114 views

E. coli values for [enzymes], [metabolites] and kinetic rates

In my attempt to create a metabolic model of E. coli, I have found a comprehensive list of metabolic reactions and their stoichiometry. The one I am currently using is E. coli model iJO1366 (more ...
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1answer
82 views

What in soy are people allergic to and will these allergens be passed along if an animal that has eaten soy is consumed?

I have two related questions about soy allergies and possible reactions. What is it that are humans who have soy allergies actually allergic to? What is the allergen? If an animal eats soy, are ...
3
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1answer
77 views

Can I use PCR buffer instead of cDNA synthesis buffer?

I am using Fermentase First strand cDNA synthesis kit but its buffer is over. I need to work today but I have not access to any sorce currently. I have PCR buffer in the lab. Can I use PCR buffer ...
4
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1answer
169 views

Can forensic DNA analysis be used to generate a visual approximation of a suspect?

In light of the current US supreme court case, I'm curious if enough information can be teased out of a DNA sample to get a "reasonable" approximation of the suspect (never mind the legality). I ...
6
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1answer
370 views

How does Topoisomerase II inhibition affect cancer cells?

Topoisomerase II poisons represent some of the most important and widely prescribed anticancer drugs currently in clinical use. These drugs encompass a diverse group of natural and synthetic ...
3
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1answer
477 views

Difference between “transcriptional activity” and “RNA expression”

I am working with a network object where the nodes are different events in molecular biology. Specifically; Catalytic activity of X, RNA expression of X, GTP-bound activity of X, Kinase activity of ...
9
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2answers
5k views

What is the difference between SOLiD, 454, and Illumina next-gen sequencing?

I've started teaching myself about next-generation sequencing in preparation for a new job, and I'm wondering what the main differences are between the 454, SOLiD, and Illumina/Solexa machines, in ...
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2answers
203 views

Question about equilibrium potential formula

My biology book says, that the equilibrium potential for an ion with a charge of +1 is: $$E_{ion}= 62mV \biggl(\log\frac{[ion]_{outside}}{[ion]_{inside}}\biggr)$$ Where does the 62 mV come from? How ...
4
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1answer
244 views

Why am I getting low transformation efficiency with DB3.1 E.coli cells?

I am making competent cells using DB3.1 E. coli cells. Even after following the exact protocol (Inoue method for ultracompetent cells) given in 'Sambrook and Russel', I am not getting transformation ...