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

learn more… | top users | synonyms

4
votes
1answer
65 views

Protein Isolation from mammalian cells

What non-SDS buffer can I use to isolate protein from mammalian cells? The protein is intended for testing electron transport chain complexes.
4
votes
3answers
177 views

Western Blot: Fischer ECL substrate 2:1 instead of 1:1?

i recently developed a b-actin-ab-incubated PVDF-WB-Membrane with the Thermo Scientific Pierce ECL Kit. According to the manufacterers orders i mixed Reagent 1 and Reagent 2 1:1. But unfortunately i ...
4
votes
1answer
452 views

What is a protective epitope?

What is a protective epitope? An epitope is basically a part of antigen. So does it mean that when the epitope combines with an antibody, it helps in the functioning of the antibody instead of going ...
4
votes
2answers
384 views

Signaling through G protein Coupled Receptors?

There are two different cell lines but we do not know that these cell lines have Gs or Gi proteins, associated with their G-protein coupled receptors. If we wants to know about this. Can we design a ...
4
votes
2answers
121 views

In DNA repair, how is it determined which strand contains the error?

DNA replication is more accurate than transcription (or RNA replication) because mechanisms exist for proof-reading and repair of DNA, but not for RNA. Consider a segment of a DNA strand, AGTC. Its ...
4
votes
2answers
112 views

Is it possible to create a restriction map using python? [closed]

I have some fragments of DNA from single and double digests using three different restriction enzymes. I'm trying to construct a restriction map of a linear fragment of DNA. The map needs indicate the ...
4
votes
1answer
87 views

Negative feedback loop and oscillations

According to the textbook Alberts Molecular Biology of the Cell (5th ed., p. 902), negative feedback loops cause oscillations when they are long delayed. I just can't figure out why. Except for that,...
4
votes
1answer
178 views

How does NHEJ cause indels?

I was reading up on CRISPR-cas9 and how it works and I am having trouble wrapping my head around how NHEJ to repair the DSB can cause indels to occur. Shouldn't the NHEJ just stick the two strands of ...
4
votes
1answer
238 views

Why do some protocols require prewarming a liquid medium before inoculating?

For example, in this protocol for E. coli competent cell preparation, it says: Plate 10 uL E. coli BL21(DE3) cells on a LB-agar plate; incubate overnight (12 hours). Prepare 500 mL SOB medium ...
4
votes
2answers
43 views

Transformation efficiency

I accidentally spun down the cells after heat shock treatment before adding the media into the tubes while doing transformation? will the transformation work?
4
votes
2answers
162 views

Redundancy of the genetic code

One particular codon codes only for one amino acid, but an amino acid can be coded for by several different codons. Now according to the genetic code, the codon UUU ...
4
votes
1answer
90 views

Influence of temperature on transcription, protein binding and decay rates

I am the kind of biologist who doesn't know much about molecular genetics and about the dynamic of biochemical reactions. Question My question concerns the influence of temperature on the dynamic of ...
4
votes
1answer
128 views

Topology of closed circular DNA

Why are covalently closed circular plasmid DNAs naturally found in an underwound state ? Is it because this makes it easier for the DNA replication machinery to access and unwind DNA ? Or is it ...
4
votes
1answer
48 views

A good software for agent based modelling?

Hello i'm gonna start my master degree in systems biology and i'm thinking of working with agent based modelling, but i really don´t know which platform can be the best to simulate interactions ...
4
votes
1answer
51 views

Is HSV-vector-mediated miRNA expression in dorsal root ganglia stable?

My question is on the following article: "Reduction of voltage gated sodium channel protein in DRG by vector mediated miRNA reduces pain in rats with painful diabetic neuropathy" My question is, do ...
4
votes
2answers
92 views

How can (or did) Deinococcus radiodurans continue to evolve after developing resistance to mutation?

Deinococcus radiodurans has a remarkable ability to resist damage to its DNA due to radiation, dehydration or (to my knowledge) any other source. It keeps multiple copies of its genome and has a ...
4
votes
1answer
102 views

How is the type of genetic manipulation determined in CRISPR-Cas9?

I've been reading up a bit on the CRISPR-Cas9 system for gene manipulation. From what I read, it introduces double-strand breaks at specific points determined by the choice of sgRNA. But how do you ...
4
votes
1answer
65 views

What's up with all the vague protocols? [closed]

I have lost count of how many protocols I've seen, including those supposed to be professionally written (such as manuals that come with kits from well known brands, or methods sections of papers in ...
4
votes
1answer
638 views

Are there any organisms that lack ATP synthase?

ATP synthase is used by cells to synthesise ATP from ADP using energy from a proton gradient. Depending on which sites I look at, this is either a universal feature of living cells, or it's "almost ...
4
votes
1answer
125 views

Ribosomal RNA amount in a Drosophila cell

I am isolation RNA from Drosophila larvae brain with TRIzol method. What percentage of extracted RNA will be ribosomal RNA? I am only interested in mRNA, so I am trying to figure out whether I need to ...
4
votes
1answer
726 views

DNA content in plant seeds vs. fruit flesh

Is there a publication comparing DNA yield and/or PCR-amplifiability after extraction from fruit flesh (like apples, oranges, cherries etc) in comparison to seeds of the same fruits? I would prefer a ...
4
votes
1answer
644 views

Effect of single nucleotide deletion or insertion on primer annealing

How is primer annealing, and, consequently, PCR amplification affected by single nucleotide deletion or insertion inside the primer ? Imagine a primer like this: GCGTCATAAAGGGGACGTG (primer) and ...
4
votes
2answers
302 views

Is AT or GC content important in electrophoresis?

Will it make a difference in running speed if we run samples of same no of bases but different AT - GC content ?
4
votes
2answers
218 views

Randomness in living systems

The point of my question is not to talk about events that are uncontrolled by living organisms. My question is about controlled randomness, or I'd like to say adaptive random process. Process that are ...
4
votes
2answers
800 views

Does bleach destroy RNAse activity, and if so, how does it do it?

I am working with RNA samples, and I'm trying to be very careful about RNAse contamination. I have some questions about bleach, though. Some people say that a solution of bleach is enough to destroy ...
4
votes
1answer
60 views

Directed evolution: Point mutation vs Insertion-Deletion vs Shuffling

When attempting enzyme function improvement via Directed Evolution I can see three different strategies to generating variation for the gene sequence: Point mutations Insertion / Deletions Shuffling ...
4
votes
2answers
167 views

Does suffocation pain involve nociceptors?

I never suffocated myself so not entirely sure, but when you suffocate, it's painful, right? But of course different kind of pain than being injured or sick. What I'm wondering is, if the "painful" (...
4
votes
1answer
43 views

Oligonucleotide purification with desalting

I have ordered 36bp oligonucleotides that anneal to each other and create sticky ends to be cloned in a vector afterwards. I have tried cloning many times with different methods and I failed. Now I ...
4
votes
1answer
85 views

protein-binding sRNAs

I am actually a computational biologist, pardon me if my question description is a bit off-note, my curiosity is driving me on A bit of background and context: In bacteria, a regulatory protein ...
4
votes
1answer
177 views

No digested product bands, but markers are visible. What could be the reasons?

I had to digest my insert containing plasmid and another vector into which my insert has to be ligated, each with BamHI and NotI. Upon digestion, I ran the gel to check for results. The digestion ...
4
votes
1answer
104 views

Do mitochondria use ATP in order make another ATP from glucose?

Do mitochondria use ATP in order make another ATP from glucose, or does it use another source of energy to convert glucose to ATP?
4
votes
1answer
604 views

RNA isolation from Drosophila head

I need to isolate RNA from Drosophila head. I basically chop the head off and first homogenize it with a homogenizer (similar to this: http://www.omni-inc.com/omni-tissue-homogenizer-th-package-p-11....
4
votes
1answer
110 views

Large scale reverse transcription?

I need to make RNA:DNA duplexes. I can make 100 to 200 ug of mRNA through in vitro transcription, and I know how to use reverse transcription to make a cDNA library, but I have questions with this. ...
4
votes
1answer
1k views

How to secrete a recombinant protein from E. coli?

What are some secretory pathways that can be used to secrete a recombinant bacterial enzyme from E. coli? I have a recombinant protein (29kDa) that I will express in E. coli BL21 cells. For now I ...
4
votes
1answer
417 views

Can I heat Trizol?

I wonder if I can heat Trizol reagent for 30 min 65C. The goal is to disrupt protein-RNA complex while inhibiting nucleases. (I can't use RNasin cause it's inactivated in 65C, and can't use RVC cause ...
4
votes
1answer
40 views

Can you knockout specific receptors in an adult?

Sorry I don't have a good understanding of this topic, but I'm guessing that "receptor knockout" is related to/a part of "gene knockout"? And If I understand correctly, gene knockout is currently ...
4
votes
1answer
54 views

Function of NaCl in yeast shuttle prep of plasmids

What is the role of NaCl in plasmid isolation from yeast cells using plasmid rescue solution? If the basic theory behind the method is alkaline lysis, then we is NaCl used and not NaOH?
4
votes
1answer
55 views

What was Protein G named after?

Protein G (the bacterial antibody binding protein) is often used to pulldown antibodies, for example in chromatin immunoprecipitation (ChIP) experiments. However, I was unable to find a site ...
4
votes
1answer
86 views

Why is an A-U bond less stable than an A-T bond?

I have encountered the following fact many times, but have not yet encountered a possible explanation for it. Will you please help me understand the molecular mechanism by which the bond between ...
4
votes
2answers
337 views

How to Extract RNA and perform RT-qPCR from very few cells ( ~5,000)?

I am currently conducting an experiment which involves FACS-sorting a specific population of cells using an antibody of interest. In order to validate the type of cells I have collected using this ...
4
votes
1answer
237 views

blue/white screening - results are opposite as expected

We inserted GFP-gene into plasmid Bluescript SK+, transformed E. coli with this construct, and then plated on an agar plate with Ampicillin and X-gal to do a blue/white screening. We got blue colonies ...
4
votes
1answer
46 views

A few questions regarding immunology [closed]

I know that there is a variable region on antibodies which can recognize a wide variety of antigens, and that germinal centers create more "fit" antibodies to respond to an infection. So I was just ...
4
votes
1answer
509 views

How much does the Hayflick limit/telomere length vary across taxa and within humans?

Since they seem to be quite connected, I'm curious if anyone knows of research comparing the Hayflick limit (and presumably by extension telomere length) between different taxa. I've heard the ...
4
votes
1answer
265 views

Cloning two fluorescent proteins with different promoters in the same plasmid?

I want to clone two fluorescent proteins both driven by different promoters in a plasmid to be used to transform B. subtilis at the amyE locus. For this I want to use plasmid pSG1729 (Lewis and ...
4
votes
1answer
73 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?
4
votes
1answer
395 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 ...
4
votes
1answer
528 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 ...
4
votes
1answer
54 views

How to find ion/water channel related genes

We now have a collection of transcripts at hand. We would like to investigate some particular ones, which are ion/water channel related. How to perform this? Could anybody point out how to find the ...
4
votes
0answers
79 views

Does Cas9 require a nuclear localization signal (NLS) for it to work in a Eukaryote?

I'm trying to establish if it's required to add a NLS to Cas9 when expressed (or transfected) in a Eukaryotic cell. Several papers report using a viral NLS, but is it absolutely necessary? Could ...
4
votes
0answers
41 views

Does depolymerisation take place at the minus end of microtubule?

Wikipedia says that Dynamic instability refers to the coexistence of assembly and disassembly at the 'ends' of a microtubule. but Karp's Cell Biology, 7th edition says Dynamic instability ...