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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I accidentally plated DH5a E. coli and left them in the 4℃ for 12 hours, if I put them into the 37℃ incubator will they start growing?
I started ligation of my constructs late last night and was super tired, since I used all of my ligation mixture I would rather not have to re-ligate.
I did my transformation procedure as follows:
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How to separate two plasmids from E coli with the same backbone?
I'm using transformation-associated recombination (TAR) in yeast to capture a biosynthetic gene cluster (32 kb) by transforming gDNA and a capture vector (11 kb) with homology arms. I identified a ...
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Griffith's Experiment Conclusions
My teacher gave us these two questions on Griffith's experiment.
How did Griffith's experiment rule out that the rough cells could have used the capsules of the smooth cells to become pathogenic?
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Terminology for Transgenes
I'm designing an in vivo delivery vector for therapeutic transgenes.
I have two different potential versions of the transgenes. If they were innate, they'd be referred to as "alleles." Does ...
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Plasmid Design and Integration events [Single vs Double cross over]
When linearising a vector by restriction digest within the middle of a homologous region can a single cross over integration event only occur if the plasmid is re-ligated within the cell after ...
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In traditional plant cloning, why do we require two different vectors (plasmids)?
So I was recently taught cloning in plants and I came to wonder what is the need to first put the gene of interest in the entry vector plasmid and then the final vector plasmid before finally ...
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How much Kanamycin is too much for selection in dh5α E. coli?
I'm using Kanamycin for selection after electroporation of my plasmid containing a kanamycin-resistance gene at a concentration of 50mg/L prepared in 1000x stock that is usually kept in frozen ...
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Which method of gene amplification for toehold switches?
My team and I are from a high school and are planning to carry out some research investigating some toehold switch riboregulators which we have designed in silico. However, we have little experience ...
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How to identify the GPD gene when the sequence varies between organisms?
I'm reading a paper on genetic transformation of a fungi and the plasmid used in the paper uses two forms of the same GPD (glyceraldehyde3-phosphate dehydrogenase) promoter to drive a GFP gene, one ...
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Questions regarding transformation in bacterial cells
First off, in transformation the donor DNA aligns itself with the complementary bases in the recipient DNA.
Now a "perfect" alignment of the donor DNA ( Sorry if my terminologies are ...
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Plating 96-well bacterial transformations
I need to clone 96 different inserts into the same backbone in arrayed format. I am planning to perform Gibson assembly reactions in a 96 well plate, and then do chemical transformations in a 96 well ...
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Gene of interest number of copies in transformed K. pastoris
When transforming a K. pastoris strain, is there a way to predict how many copies of the gene will be integrated following the homologous recombination? More specifically is it something that a ...
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How does the DNA cross through bacterial cell wall during electroporation?
There exists a lot of literature on electroporation of Gram-positive and negative bacteria. Most of it gives an explanation that electroporation works by creating transient pores in cell membranes of ...
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Is a butterfly one or two animals?
I have read somewhere that a butterfly might be two animals that combined together. One animal was a worm-like creature and the other an insect.
And the insect basically hatched inside the worm. ...
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How can I improve efficiency of Ecoli transformation?
I am an intern in biology institute. I have a 17.3kbp plasmid need to transform to Ecoli. But I have tried many time but have no or very few colony on LB plate.
I know large plasmid have less ...
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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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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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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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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 ...
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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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