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

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7
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0answers
147 views

What is the fastest way to build an alanine scanning library?

For interfacial studies, I would like to build an alanine scanning library for one of my proteins examining 20 sites. I will ultimately express the gene using E.coli cell-free protein synthesis. I ...
9
votes
1answer
228 views

How are there alternative initiation codons?

According to wikipedia and the original complete sequence of the K-12 genome, there are multiple non-AUG start codons such as GUG and UUG. How is this possible? I'm particularly curious about the ...
6
votes
1answer
239 views

Effect of histidine on the binding affinity of HisP

I was asked the following question by my teacher: A gene regulatory protein called HisP regulates the enzymes for histidine biosynthesis in the bacterium E. Coli. HisP is a protein whoes ...
9
votes
2answers
15k views

Why is SOC medium recommended for transformations?

In pretty much every transformation protocol I've seen SOC medium is used to grow the bacteria for a short while after the tranformation and before plating. I've usually substituted LB medium for ...
7
votes
1answer
117 views

Does the MS2 RNA binding protein have any translational repression effects?

Reposted from Quora: http://www.quora.com/Does-the-MS2-RNA-binding-protein-have-any-translational-repression-effects I'm thinking of the MS2 protein binding to its RNA hairpin target. Would the ...
0
votes
1answer
79 views

If inhibiting S6 kinase decreases protein translation, then could inhibiting S6 kinase could possibly slow down long-term potentiation in neurons?

From http://en.wikipedia.org/wiki/P70S6_kinase... Phosphorylation of S6 induces protein synthesis at the ribosome. P70S6 kinase is in a signaling pathway that includes mTOR (the mammalian ...
5
votes
1answer
80 views

Why would we overexpress Sir2 by overexpressing its hypomorph (dSir2-EP2300) in C. elegans?

Can't we just overexpress regular Sir2 in the paper? Rather than overexpress a reduced-function gene? The paper is Burnett C, Valentini S, Cabreiro F, Goss M, Somogyvári M, Piper MD, Hoddinott M, ...
8
votes
1answer
109 views

Why is propanoic acid often added to the diet of C. elegans?

And what are its effects on C. elegans? The paper is Burnett C, Valentini S, Cabreiro F, Goss M, Somogyvári M, Piper MD, Hoddinott M, Sutphin GL, Leko V, McElwee JJ, et al.. 2011. Absence of effects ...
9
votes
3answers
1k views

Why some genes are dominant to each other? What is the mechanism behind it?

If i have a brown eye gene which encodes the protein that is responsible for the brown color, and have a blue eye gene either, what is the reason that my eye color is brown? How does one gene ...
19
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1answer
398 views

Regulation of chromatin structure

Recently, I reviewed the different levels of chromatin structure. The primary level is nucleosomes, where DNA is bound to histones, and has structural similarity to "beads on a string." The secondary ...
9
votes
2answers
155 views

What is the least costly method to generate sequential amino acid deletions?

I'm looking to generation sequential deletions from a gene of interest. The total size of this region is 8 amino acids. I'm trying to determine which portion of this region is necessary within the ...
10
votes
3answers
2k views

How long can E. coli stocks be stored at -20°C?

I'm volunteering for a biohacker lab - biocurious in Sunnyvale. The have a pretty good set of equipment - gel boxes, incubators, but they don't have a -80°C freezer yet. I'd like to set up some ...
7
votes
1answer
128 views

PDB Mining: Why Do I Find Atoms Less than 1 Angstrom Apart?

I am attempting to find potential Hydrogen bonds between Hydrogen donors and aromatic ring acceptors. I do this by predicting the location of Hydrogens on residues and then calculating how far these ...
16
votes
1answer
683 views

ATP cost for gene expression

How would you estimate the number of ATPs required to transcribe, export and translate a single eukariotic protein?
17
votes
3answers
993 views

How long can I store extracted RNA?

If I extract RNA from a (leaf tissue) sample using a one-step phenol:chloroform extraction, how long can those samples be stored at -80°C? And how many times can I defrost and refreeze them before ...
9
votes
2answers
2k views

How do I clean and calibrate pipettes, and how often should I do it?

I work in a lab where all the pipettes are shared. We often have visiting students who come and use the pipettes for a short project. So when I work with them, they might have been handled by other ...
10
votes
1answer
18k views

Why do we add salt when precipitating DNA?

All the DNA extraction protocols I have seen involve adding salts to the extraction buffer. What is the purpose of the salts? What happens if they aren't included?
10
votes
2answers
4k views

How do I clean phenol contaminated RNA without losing any of the sample?

I recently extracted RNA from developing plant leaves for the first time, as part of a very long and intensive experiment. The samples were extremely precious because of the amount of effort that went ...
7
votes
1answer
397 views

How do you knockout an E. coli gene without disrupting the rest of the gene cluster?

I'm familiar with the method to build Keio collection using single-gene deletion with an antibiotic cassette. However, what happens when there are gene clusters or overlapping ORFs and you still only ...
16
votes
3answers
1k views

How and where, in the human brain, are memories stored?

Background I am a computer programmer who is fascinated by artificial intelligence and artificial neural networks, and I am becoming more curious about how biological neural networks work. Context ...
2
votes
1answer
82 views

Is the protein in teardrops still attached to cells, or is it released and free-flowing?

A ScienceDaily article says that the protein in teardrops can kill bacteria. But how does it reach the bacteria?
13
votes
1answer
195 views

What is the prehistory of amino acids in cells?

As a followup to Why 20 amino acids instead of 64? and What is the smallest number of amino acids required for life?, I am trying to understand the prehistory of amino acids in cells. All living ...
6
votes
2answers
734 views

Reverse transcription PCR optimization

What is the ideal amount of RNA to use for the RT? and how much cDNA to use then for the PCR? I did RT with a solution of RNA of 0.36 ug/ul. Then for my PCR I used 1 ul of the cDNA obtained and used ...
9
votes
1answer
24k views

Absorption ratios 260/280 and 260/230 for RNA

I extracted RNA from different cell lines, an I want to perform reverse transcription and then PCR. To get good results, in which range should the absorption ratios 260/280 nm and 260/230 nm be? And ...
13
votes
2answers
475 views

Intrinsically disordered proteins as potential drug targets

Intrinsically disordered proteins (IDPs) are a class of proteins that do not adopt a stable secondary or tertiary structure under physiological conditions in vitro, but still have biological ...
7
votes
1answer
2k views

The effect of the start codon GTG on translation in E. coli

Translation in E. coli is initiated at the start ATG codon, which encodes for the amino acid Methionine (Met). In some cases, the start codon can be GTG, which within the open reading frame (ORF) ...
5
votes
1answer
99 views

What are the different ways an exon gets spliced?

Exons are produced by more than one mechanism, e.g. splicing out introns after transcription, if I remember correctly. Please list all mechanisms.
27
votes
4answers
545 views

Why are amino acids in biology homochiral?

Why are nearly all amino acids in organisms left-handed (exception is glycine which has no isomer) when abiotic samples typical have an even mix of left- and right-handed molecules?
20
votes
1answer
505 views

How, on a physical level, does ATP confer energy?

When ATP is used as the energy currency to make, say, reaction X + Y → Z happen, is what happens on a physical level down at the molecular scale that during the reaction ATP + H2O → ADP + Pi ...
6
votes
1answer
201 views

In C. elegans, why does knock-down of cco-1 in some tissues increase lifespan, and knock-down of cco-1 in other tissues decrease lifespan?

Full question: In C. elegans, why does knock-down of cco-1 via RNA interference in specific tissues like body wall muscle decreases life span, whereas knock-down in the nervous system and intestine ...
10
votes
1answer
344 views

What is the functional and structural distinction between core (H2A, H2B, H3,H4) and linker(H1/H5) histones?

Many explanations of histone biochemistry isn't quite elucidating for the undergraduate student. How does histone structure (dimers, octomers) relate to their specific functions as core or linker ...
7
votes
1answer
749 views

What is the mechanism of transgene integration (from expression vector to the host genome)?

How does a transgene (in vector) integrate to the host genome? (e.g. in glass bead method, neither biolistic nor agrobacterium). I already cut some parts (NdeI-PciI) of the vector (pUC18) and ...
10
votes
2answers
758 views

Why are restriction enzymes not frozen?

We all know restriction enzymes are proteins, but we never freeze them. They are instead provided in high glycerol containing solutions by companies and stored at -20C. Is there a reason why this is ...
14
votes
6answers
1k views

Online Molecular and Cellular Biology Video Lectures?

I am looking for video lectures to go through to guide my reading in intro molecular and cellular biology. I've had intro bio and I study evolutionary theory, but my molecule- and cell-level knowledge ...
23
votes
4answers
517 views

How are the boundaries of a gene determined?

What statistical processes and methods are used by geneticists/molecular biologists to know where one gene starts and one ends?
9
votes
2answers
531 views

What is causing my problem with very low yields when isolating a 42kb yeast plasmid?

I have to isolate a large plasmid from yeast and transform it in E. coli. After transformation, I often get no colonies. One reason for that is the yeast mini prep hasn't worked or the DNA ...
5
votes
4answers
1k views

What are good resources for self-learning modern molecular biology concepts? [closed]

My learning of molecular biology ended in the early 90's (and with early 90's era information). While I don't aspire to be a molecular biologist, I do aspire to better understand modern approaches ...
6
votes
2answers
774 views

How do I prepare and clone from E. coli DNA?

I'm looking for a protocol to get genomic DNA from an E. coli sample so that I can clone a small portion of it using PCR into a plasmid. (< 500 bp in this case). It seems OWW (Open Wet Ware) ...
9
votes
1answer
773 views

Can I elute my GFP-tagged protein off anti-GFP antibody using a peptide?

I would like to perform a modified co-immunoprecipitation assay using a GFP-tagged protein. We are going to bind the tagged protein to anti-GFP antibody then bind that to protein A/G beads, however ...
10
votes
1answer
916 views

How is Taq polymerase produced?

I've seen Taq polymerase being marketed as either "native" or "recombinant". I understand that the recombinant version is produced by specially modified Escherichia coli strains that have the gene for ...
9
votes
2answers
193 views

bi-directional transcription experiment

We suspect a bi-directional transcription event is happening at a locus in our organism where two genes are directly adjacent to each other. The annotation data is not well established. The intergenic ...
4
votes
5answers
488 views

What is a good miniprep protocol for the class room?

I'm trying to find a good protocol for plasmid minipreps and I'm looking at 3 preps I've found: Using phenol/chloroform extract with phenol:chloroform:isoamylalcohol, isopropanol precipitation, ...
8
votes
2answers
446 views

Has anyone tried Gibson Assembly Optimizations?

Has anyone tried the chew back-anneal in vitro DNA assembly method (known as Gibson Assembly) for difficult sequences, like GC-rich sequences? How big constructs could you efficiently assemble? Did ...
9
votes
4answers
207 views

How can I avoid digesting protein-bound DNA?

I'm interested in sequencing and analyzing the bound DNA, and minimizing the amount of unbound DNA that gets sequenced through digestion. When digesting protein-bound DNA, is all of the unbound DNA ...