- DNA technology questions
- Gel electrophoresis
- Polymerase chain reaction (PCR)
- DNA libraries & generating cDNA
- DNA cloning and recombinant DNA
- Hybridization (microarray)
- Expressing cloned genes
- Southern blot
- DNA sequencing
- Gene expression and function
- Applications of DNA technologies
- Safety and ethics of DNA technologies
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- Isnt it that the complementary DNA is a ssDNA and prior to adding it to the vector (plasmid) we first need to make a dsDNA via DNA polymerase?(18 votes)
- This seems similar to the DNA Cloning video, with the exception that in this video, they talk about getting the mRNA of the gene of interest, making it into cDNA without the introns that were cut in pre-mRNA processing (rather than using restriction enzymes to "cut" DNA from the strand).
Are these two different ways of doing the experiment that yield to the same final product of bacteria that will express the cloned gene? Or is there a difference in how the bacteria will express the gene? And does the exclusion of DNA that yielded the introns in this video matter (in terms of gene regulation/expression)?(6 votes)
- Where does the initially isolated mRNA (at 0.36) come from? Is the gene of interest used as a template to transcribe it?(3 votes)
- Good question! It's actually possible to extract mRNA directly from a cell, and that's probably what would be done in this case.
You're right that mRNA can be made by in vitro transcription, using a DNA template. However, if you're trying to isolate RNA for cloning, it's usually because you don't yet have a DNA corresponding to the RNA sequence (for instance, because the genomic DNA contains introns that must be spliced out to make the mature mRNA). So, you would want to get the RNA directly from the cell, where it's already undergone processing.(5 votes)
- could not the cDNA also be amplified with PCR? I am aware that doing so obviates the possibility of the bacteria using their own ribosomes to express the gene of interest.(4 votes)
- How is this process different from creating a DNA Library?(2 votes)
- The real difference is in the objective. Broadly, the object of cloning is to assemble a plasmid construct including some gene of interest and various promoter/enhancer/regulatory/labeling/etc. elements, and then to amplify it. The object of the DNA library is the creation of a database of proteins referenced to their respective genetic sequences. The generation of a DNA library (especially if you're working backwards from the amino acid sequence as shown in the other video) involves several cloning techniques. But by and large, the object of these techniques isn't to amplify a construct, it's to facilitate sequencing your cDNA.(2 votes)
- At1:45, the message box reads, "Here, Ronald means that you 'insert' the cDNA into a plasmid. The words 'transform' is actually used for the transfer of the plasmid into a bacterium."
According to the Kaplan MCAT book, "transformation results from the integration of foreign genetic material into the host genome."
So does "transformation" officially occur when the plasmid is inserted into the host genome? Or just when the plasmid is inserted into the bacterium (and not necessarily incorporated into the bacterium's genome)?(2 votes)
- Late answer here but transformation occurs when foreign genetic material is taken into the bacterial environment. The important note here is that transformation can result in either a plasmid DNA or DNA incorporated into the host genome. Remember that a plasmid is non-chromosomal DNA that is NOT incorporated into the host's genetic material.
I hope that answers your question!(2 votes)
- can the microarray technique be useful in the qualitative analysis of the recombinant DNA ?(if we have grown the culture which contains the recombinant DNA and we have also done the blue-white screening and grown the culture in antibiotic media to separate the simple transformant and recombinant cell. and we want to know if there is any false expression in the white colony or not)(1 vote)
- [Voiceover] Normally when we think about cloning, we think about cloning in this sense. So let's say that we've got a baby and it's just so cute that we want two of them. So we can go and clone a baby. Well this isn't exactly what we're talking about when we talk about cloning in a scientific sense. Normally what that means, scientifically is let's say that I've got a cell and there's a certain gene, let's call it gene A for now, and I want to clone that gene. So how do I go about cloning gene A? Well, the first step is to isolate the messenger RNA from the gene of interest. So if we have the messenger RNA, what we need to do is we need to convert it into DNA. So if we add reverse transcriptase, which is an enzyme that will go and actually create DNA from RNA we'll end up with something known as complimentary DNA. Now complimentary DNA, and that's what the C stands for, is basically the complimentary DNA sequence to the MRNA sequence. Now keep in mind that the complimentary DNA only contains exons, so the introns have already been spliced out. We talk about introns and exons in another video. So next, what we have to do is we take this CDNA, this complimentary DNA, and now what we want to do is amplify it. We want to create lots and lots of the complimentary DNA so that we can have plenty of CDNA to work with. In order to do that, we have to take this complimentary CDNA and transform it into a plasmid. Now, what does transform mean? What it basically means is that we're taking the complimentary DNA and we're basically putting it into a plasmid. So we're gonna transform it into a plasmid and this plasmid is going to contain antibiotic-resistant genes. Antibiotic-resistant genes. We'll talk about why that's important in just a second. Now that we have this plasmid, we want to actually infect bacteria with the plasmid. So we want to put it into bacteria. Once it's in the bacteria, what we want to do is we want to actually add antibiotics to the cultured bacteria. So we're gonna add antibiotics and since the plasmid contained antibiotic-resistant genes, the bacteria that were successfully transfected will survive and all the other ones that didn't have the plasmid inserted will die. So now we've got pure cultured bacteria that all contain the plasmid which contains the gene of interest, the DNA of interest, and now the bacteria just does its thing so it's just gonna start to replicate. The bacteria is gonna replicate and as it's replicating, it's gonna produce lots and lots of the gene of interest. It's gonna produce lots of the MRNA of interest. Basically what we just did through this process is we just cloned the gene of interest. And that's what we're talking about when we talk about cloning, at least from a scientific point of view.