Tag Info

New answers tagged

2

It depends on the seed and fruit you're talking about. The amount of DNA in your crushed up sample of plant matter depends on only one thing--how many cells are in your sample. Look at this diagram of a kernel of corn (U of Indiana): There are different cell densities in each area--in the endosperm, the cells can be huge (and therefore have a lower ...


2

What you can do depends on your proteins: If both proteins (your target protein and the loading control) are seperated far enough, you can detect both of them in the same step by adding both primary antibodies and both secondary ABs into the buffer at the same time. They will bind only to their specific epitope and you will get nice signals. This is ...


10

You can validate the interactions by knockding down (KD) or overexpressing (OE) a gene and checking the change in expression levels of the downstream nodes. You can do this in a high throughput fashion using microarray or RNAseq. For protein you can do an LC-MS. However this method cannot help you in: Differentiating direct vs indirect interactions Finding ...


3

Just to add to the above responses, you are better off not use MQ because if it is contaminated with DNases, then it will digest your construct if it reaches room temperature or 37 oC as you are melting the frozen construct in your hand! TE-buffer is the standard buffer for DNA elusions but make sure your column is compatible with it. For transfection based ...


5

The Quiagen kits I'm familiar with explicitly state that you can use water instead of elution buffer for elution. The choice of continuing to use buffer or water is yours and depends on what you want to do with the DNA and whether the buffer interferes with later steps. Using the buffer is probably safer for long-term storage, but I don't know how much this ...


6

I would generally recommend using the elution buffer (which is typically Tris-EDTA buffer) or TE-Buffer, as the pH and the conditions stabilize the DNA for a longer time, which is especially interesting when you want to store your DNA. It also dependent on your downstream application - when they recommend diluting the sample in water (or another buffer) I ...


0

Rosetta strain is normally used when people try to expression mammalian genes in E. coli. Origami™ strain is used for protein folding. you may need triple antibiotics to select your clones. This may impede bacterial growth.


3

This is too long for a comment, so I post my thoughts about this here: This falls into different important parts here: One is an insufficient understanding of the techniques on which the method is based. This makes it hard to decide which steps are important and which are not. One prominent example would be the use of EDTA in DNA gel running buffers. This ...


2

it depends on which PCR polymerase you're using. When doing some previous methods development, I tested about half a dozen polymerases amplifying either directly on the beads or after elution. Some polymerases worked equally well under either condition, while others were completely inhibited by the presence of beads. However, elution from the beads is ...


1

6kDa are usually enough to seperate two proteins in my experience. Try the other way, use a 7 or even 8% gel. They run longer but have a higher separation capacity. If you look at this figure from the LabFaQ, you see that you can use a higher percentage gel: The other thing are the antibodies: Where are your isoforms different? 6kDa are 55 to 60 amino ...


0

Methylation played different roles for different types of organisms throughout evolution. For bacteria, methylation protects against self-digestion of DNA by restriction enzymes. In eukaryotes, methylation assumes many different roles (e.g., mutation-rate regulator, gene expression regulator, chromatin structure regulator, etc.). Insects have very little ...


1

If the gene is supposed to be well conserved then you can use the sequence from the related species. For protein coding genes target the ISH probe to the coding region which is more likely to be conserved. What you can also do subsequently is to: amplify the CDS do 3' and 5' RACE Clone in a plasmid (do a blunt end or TA cloning) Sequence the insert ...


1

This is a pretty broad question, but I will try to give an answer: This process will undergo further evolution over time, no question. It has evolved into the current state and there is no reason to assume that this ended. When there are mutations which prove advantageous, they will evolve. This may also involve a faster response time for example, but we ...


1

Why do a gel purification? you can just use a kit similar to QIAGEN gel extraction kit and do a non gel based plasmid purification and elution, using the buffers used as usual! PCR DNA clean up kits will do as well!


3

I am sure you have done it; just a note to others: do a PCR screen to ascertain that the plasmid is there. For minipreps you generally do not need a starter culture. If you have screened your clones and now just want to amplify and keep the plasmid for cloning purposes then go for a maxiprep with 200-400ml cultures. Do not overgrow the cells, your ...


2

Can't exactly say what problem you are facing but you can follow these steps for efficient restriction digestion and elution. Never set up a digestion reaction with more than 1µg DNA per 20µl; if you require more digested DNA, then set up multiple 20µl reactions with maximum of 1µg DNA in each (500-700ng is ideal). 10µl enzyme for a 230µl reaction: bad ...


1

It is possible to tag molecular motors with fluorescent proteins. It may impede with its movement but as this paper describes, a variant can be created that doesn't have cargo binding ability. Constitutively active kinesin motors can be generated by truncations that remove autoinhibitory and cargo-binding regions of the polypeptide. For this work, ...


1

From this paper: We chose to test for Cas9-driven targeted mutagenesis on the endogenous Trp53 gene in mouse embryonic fibroblasts (MEFs), a cell line in which the biology of p53 has been thoroughly characterized and where the gene is structurally intact. Furthermore: Seventy-two hours post-transduction, cells were cultured in the presence ...


0

Pretreat the enzyme at high temperature before carrying out the assay at the standard temperature – Alan Boyd


1

There are always exceptions but you can consider some general rules. A is 1 i.e the sequence that is perfectly complementary (2 is complementary but direction is parallel — cannot base-pair), then B would be 1 (siRNA). Reasons (some are just based on general rules of siRNA design): siRNAs have to be perfectly complementary The size is also perfect for a ...


1

It doesn't seem like a homogenization problem. Nonetheless you can try this: Suspend the heads in trizol Homogenize by passing through 30/31G needles (insulin syringe) up to 10-15 times. Heating a little might help but I think it is unnecessary for these tissues. Insulin syringe is much better than the hand held homogenizer. Freeze-thaw 1-2 times if you ...


4

This is not as hard, as it first seems. Lets have a look at the single enzyme digests first: The digest with enzyme A and B only leads to products which are 5kB (5000 bp) away from each other. Since they are of the same size, both equally sized restriction fragments appear as one band. So each enzyme cuts the plasmid exactly in half. The double digest is a ...


8

Hydrogen peroxide as a reactive oxygen species (ROS) is a highly problematic molecule for the processes inside of cells (it can spontaneously oxidize and damage cellular components). With Catalase, organisms have a very effective enzyme which degrades the peroxide into water and oxygen. However, although the molecule is problematic, it is still produced by ...


8

Actually, there's a lot of animals, plants and microbes can do it. However, it is just an Intermediate process in general. Here's a strong example. In the condition with the absence of catalase, glucose oxidase can catalyze glucose + H2O + O2 become gluconic acid + H2O2. Another process can also generate H2O2 catalyzed by Ero1.


2

The best way for scaling up these kinds of reactions is to set up many reactions. Prepare a mastermix for lets say 20 reactions. You can pool them together, precipitate the RNA and dissolve to appropriate concentration in nuclease free water. Also, use gene specific primer instead of oligo-dT and don't add poly-A tails (poly-A does not affect the in-vitro ...


0

Actin is required for the neurite architecture and is present throughout the shaft (along the direction of the neurite process) See these images.                                                           ...


2

This is a general answer for all three of your related questions: This one How does Temperature influences the rate of protein turnover? How is the rate of transcription influenced by temperature? Since you said: I want to simulate the evolution of genetic architecture when after a sudden change in temperature or in an environment that is ...



Top 50 recent answers are included