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Questions tagged [transformation]

The genetic alteration of a cell resulting from the direct uptake, incorporation, and expression of exogenous genetic material from its surroundings.

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are there any species that we can identify as being 'mid-way'though an evolutionary change?

I have just learned that dolphins evolved from a dog/cat like land mammal (Mesonix) that became ever-more water venturing. I understand and can visualise how the arms legs and tail slowly evolved into ...
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1answer
246 views

Heat shock vs electroporation

I've been transforming E. coli via heat shock in order to insert oligonucleotides (around 50 nt); however, none of my experiments have given positive results so far. I begin to question the efficiency ...
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37 views

What are the advantages using SUMO vector for expression? [closed]

My question is : What are the advantages using SUMO vector for expression?
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84 views

Yeast transformation plate confluent with growth

I have problem with yeast transformation. I tried to transform pyes3-tryp(my insert inside) into INVsc1 yeast cell by following the protocol in the manual for few times but failed(confluent with ...
3
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1answer
44 views

Cloning DNA fragment - at least trying to

I`ve been trying to clone a fragment (about 1.6 kb) using pGEM system, unfortunately it was not succesful. Then, I moved to pJET, and guess what? Again, very low transformation efficiency. Now I am ...
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1answer
83 views

Why must gene entrapping constructs be integrated into introns and not exons?

Homework Question: In enhancer trapping, reporter transgenes are integrated randomly into the fly genome and their expression identifies enhancers and thereby genes expressed in particular patterns. ...
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1answer
290 views

LacZ' selection: blue colonies despite ligation of insert

It has been suggested that bacteria transformed with pBlueScript vector, containing an insert in the middle of the lacZ' gene, can still give blue colour on X-gal, if the insert is small and ligated ...
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1answer
98 views

Viruses and Transformation

Would the lysogenic cycle for the reproduction of viruses be considered a form of naturally occurring transformation since DNA from the virus is being incorporated into the DNA of the host cell?
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Luciferase promoter vector over p-AcGFP1-C1 vector

AcGFP1 vector can emit light by itself, whereas in case of Luc vector a substrate is needed for the reaction. Nevertheless, Luc is said to be more specific or better than AcGFP1. Why is this so called?...
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1answer
72 views

How to prevent a old promoter region from attaching onto a plasmid instead of a new one during ligation?

What I am trying to do is take out a existing promoter region in a plasmid, and replace it with a new one. So first I use the appropriate restriction enzymes to get rid of the existing promoter region....
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1k views

Does prokaryotic transcription have activators / enhancer regions involved?

I am designing a biosensor, and I need to know whether prokaryotic transcription involves or can involve (if a gene needs to be regulated) enhancer regions. Also, where are enhancer regions located (...
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1answer
81 views

For a recombinant pUC19 plasmid with cut sites at Hind III and EcoO109i, does lactose need to be present for the gene of interest to be expressed?

I am growing E. coli transformed with the plasmid pUC19 with an insert of an enzyme in rat brain between the mentioned cut sites (EcoO109i and HindIII). I have isolated and created the recombinant ...
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1answer
355 views

Can Bacteria Repair Dephosphorylated Plasmids?

I have been trying to insert a short sequence of DNA into a plasmid. I wanted to make two different versions, with the insert at different locations. I chose HinDIII and SalI sites approximately ...
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2k views

Electroporation vs gene guns [closed]

What are the pros and cons of using electroporators (left) and gene guns (right) for transformation in terms of: Price Target organism Efficacy Ease of use Maintenance
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1answer
368 views

Plasmid copy number and Rop protein

If i want to transform a bacteria (E. coli) with a particular plasmid (in my case pBR322) will the presence of the Rop gene affect the production of it ? Is it desirable to use a plasmid without that ...
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Has anyone tried to prepare chemically competent Mycobacteria?

Bacterial species like E. coli are easy to transform using a variety of methods, including both chemical transformation and electroporation. Chemical transformation is especially nice to use as it can ...
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9k views

Why pick just a single bacterial transformed colony

So after bacteria have been transformed to perhaps grow up a plasmid of interest, why pick only a single bacterial colony from a selective plate for further expansion? I understand that this is to ...
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2answers
1k views

Transformation of E.coli cells

As a team of undergraduate researchers we are looking to transform two genes of interest into our competent E.coli cells. Both of these genes are in separate plasmids and we would like to put them ...
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57 views

Generate T2/T1 Phage Resistant E Coli

I would like to generate T2/T1 phage resistant Stbl3 E Coli to use in virus production. Is there a plasmid somebody has used that confers resistance, or is this done another way?
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1answer
1k 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 ...
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98 views

CRISPR Knock in

Using the CRISPR/Cas9 technology, it is possible that after inducing a DSB with the Cas9 endonuclease guided with an RNA designed by the user and using a template DNA, get a desired Knock-In (KI) by ...
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0answers
56 views

What is the maximal insert length for PCR based homologous recombination in S. cerevisae

I would like to insert a 6 kbp construct, which I have on a plasmid into the genome of S. cerevisiae. This plasmid was originally constructed to integrate at the HIS locus via homologous recombination ...
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Transfect Mammalian Cells with Single Stranded DNA?

Agrobacteria can deliver parts of their DNA to plant cells as T-DNA, transfer DNA. This DNA is delivered as a single strand and can be integrated into the plant genome or can be converted to a double ...
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Can iGEM distribution parts be directly PCR'd?

The iGEM DNA Kit Plate Instructions say that there is only 2-3ng of DNA per well, which is already miniprepped and in plasmids. Then it says that "there is not enough DNA in each well to perform ...
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1answer
793 views

What is the best current understanding of how yeast transformation works?

I would like to get myself up to speed with what is currently known to science about yeast transformation. Specifically, transformation of plasmids and linear DNA fragments. I am particularly ...
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1answer
1k views

Efficiency of plasmid DNA isolation from frozen E. coli cell cultures

Has anyone isolated plasmid DNA from frozen (at -20degrees) E. coli cell cultures (not pellets)? Has that worked and if so, with what yields? What would be the quality of the isolated plasmid DNA if ...
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1answer
25k views

How does heat shock transformation work?

What exactly happens when competent cells like DH5ɑ are heatshocked with DNA present? How does the DNA get inside the cells? Specifically, why are all the steps necessary? What if you heatshock right ...
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20k views

What is the difference between transformation and transfection?

What is the difference between transformation and transfection? How do both of these methods work?
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E coli cotransformation efficiency

What is the E coli transformation efficiency for 2 plasmids? Are any studies that have looked at the correlation between number of plasmids transformed and transformation efficiency? Is there a ...
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1answer
241 views

When might an inhibitor of bacterial transformation be useful?

I am part of a project elucidating some structures that are required for bacterial transformation. We have the opportunity to screen inhibitors of the system to stop it from functioning. I am not a ...
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1answer
2k views

Problem with bacterial transformation with electroporation

I have a problem with a bacterial transformation of a yeast gene that I can not solve. I isolated yeast DNA and did a PCR to get my product. I am using pCGCUm vector with a GFP construct. I digest ...
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1answer
4k views

How to reduce/eliminate cell clumping in suspension CHO cells, without using an anti clumping agent?

what is the best way to eliminate clumping suspension cells used for transfection. Anti clumping agents interfere transfection and hence can't be used. Though maintaining the cell density low helps, ...
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1answer
658 views

How does electroporation mediated recombination work?

For yeast and other organisms, DNA can be readily taken up into the cell naturally or through membrane disruption. What does applying a voltage to cells do exactly that allows more recombination to ...
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1answer
340 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 ...
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1answer
531 views

Improving transformation efficiencies- induce supercoiling?

From my limited knowledge of science, I know transformation can be one of the hardest step in cloning, and that a large amount of research/trial and error has been done to improve on this step. I've ...
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2answers
538 views

What medical and commercial proteins are made using genetically modified animals?

Recombinant technologies in micro-organisms being used to produce commercial and medically useful proteins like insulin are fairly common. However some proteins are still produced commercially in ...
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1answer
1k views

How is the exogenous DNA protected from degradation during bacterial transformation?

During transformation, a bacterium can take up DNA from its environment. A small fraction of bacterial species are known to be naturally competent, meaning that they can engage in this sort of ...
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1answer
893 views

How the concentration of an antibiotic is chosen for YPD medium?

I need to prepare YPD plates containing hygromycin, but I don't know which concentration I can use. I'll select transformants of Hansenula polymorpha.
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2answers
2k views

Sparking during electroporation of plasmid DNA into bacterial cells

During electroporation of bacterial cells (I work with Mycobacterium tuberculosis, but I think this applies to E. coli as well), sometimes I get sparking. I've read this is due to salts present, ...
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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 transformation....
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2answers
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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 ...