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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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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37 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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18 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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23 views

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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29 views

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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123 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 ...
3
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1answer
97 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
1k 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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2answers
2k 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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257 views

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 ...
3
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1answer
93 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
633 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 ...
7
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1answer
459 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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205 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 ...
4
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193 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 ...
5
votes
1answer
174 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 ...
5
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2answers
329 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 ...
7
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1answer
508 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
136 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.
5
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2answers
564 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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6k views

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 ...
9
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19k 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 ...