Colony PCR Protocol (BIOL310)
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- čas přidán 23. 07. 2024
- In this video, I walk you through the procedure of screening bacterial colonies for your gene of interest using Colony PCR. In the process, I also introduce the concept of a PCR Master Mix, which will simplify and speed up your work.
0:00 - Introduction
0:40 - Preparing bacterial samples for use as template DNA
2:26 - Preparation of a PCR Master Mix
5:20 - Completing the PCR reaction setup
Amazing explanation thank you so much 🙏🏻🙏🏻🙏🏻
Glad it was helpful!
That's very nice
Thanks
Good explanation 👏
Thank you 🙂
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What about plasmid DNA, should we cleave the DNA with restrictase, or it directly can polymerase?
There is no need to cut the DNA - DNA polymerase doesn't need free ends on the template in order to work. In fact, we were trying to amplify a cloned gene on a plasmid here.
Also, in this experiment we were not purifying any DNA - we were using the cells directly.
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what is the DNA template used for colony PCR? (compares to a normal PCR)
The DNA inside the cells. Colony PCR simply skips the step of extracting DNA from the organism.
@@ItsLearnable isn’t that what you did when you lysed the bacteria?
@@jedn6488 Lysing the cells, is not the same as isolating the DNA away from everything else.
Thanks for the video...I am trying to learn about PCR duplication? what is it in simple terms and why it is important?
PCR is just a way of synthesizing DNA in a test tube (a very tiny PCR tube actually). It's very similar to what the cell would do to replicate it's own DNA (uses similar machinery), but we only replicate a small region - ie. a specific gene - and we do it many times over. This allows us to quickly make millions of copies of the gene of interest (it only takes a couple of hours).
@@ItsLearnable Thanks a lot....what is the reason for making millions of copies? Reading it more accurately?
@@tamer4456 Sometimes, it's as simple as just wanting enough DNA for it to be easily detectable (ie. on an agarose gel). For example, the PCR tests for COVID-19 are used to search for copies of the viral genes in a person's sample. The primers we use, generally amplify a very short sequence, we thus need many copies of such sequences for the product to be detectable on a gel (please see some sample gel images of SARS-COV2 products here: www.ncbi.nlm.nih.gov/pmc/articles/PMC7340604/figure/F2/ ). This is just an example - COVID testing usually involves the use of Real-Time PCR which has a built-in detection mechanism (no gel required) - but I hope this helps to clarify things.
@@ItsLearnable Thanks a lot also for sharing the paper.
Why should we add DNA polymerase separately?
I use this as a general rule for any enzymatic reaction. This is because the reaction will start the moment the enzyme and substrate are in the same tube (even if the conditions aren't yet what you intended). So, I always tell my students to add the enzyme last. This way you have some control over when the reaction begins, ie. all your tubes can start the reaction at about the same time.
In this case, depending on how you plan out your work and on how quickly you work, you could potentially add the Taq polymerase to the master mix. But in our case (this is an introductory molecular biology lab), it was likely that the master mix would be sitting in a tube for some time before being added to the template. So, it was simply better to have students make sure everything else is finished and ready to go before they added the Taq.
@@ItsLearnable thank you very much,
That is the best explanation 😍😍😍
Hai Sir, How long should we place the bacteria cells on ice (after heating at 94C) before centrifuge to remove the interest DNA sample?
2-3min should be enough. The purpose of this is just to get the water vapor (remember that you had just heated the sample) to condense on the side of the tube. 2-3min should be enough to time to cool things down.
Thank you sir. One more question, what about the TE buffer volume to dissolve 1 colony bacteria?
The actual volume isn't really that important (I think we used 200ul in the demonstration) - you just need some liquid to resuspended the bacteria.
Please note that whatever volume you use, make sure you measure it accurately because you will need to have an equivalent volume in a balance tube for centrifugation.
Clarifying it was Tris-EDTA buffer in the first tube?
Yes.
@@ItsLearnable Thanks! And how much of the supernatant is being added to the master mix?
@@jennaneumann8201in general, it depends on how much or how little target material you think you have in there. In our case, we used 2ul.
Since we're testing for the presence of a DNA target cloned into a plasmid, and each cell in the colony has hundreds of copies of that plasmid (we used a high-copy-number plasmid), we will potentially have lots of the target DNA there. So, you could use less than 1ul.
The reason I used 2ul is because our p20 micropipettors have a lower limit of 2ul.
Hi sir, do you use gram negative or positive bacteria?
What about gram positive bacteria? is it possible?
Sorry sir, one more question, what speed does the centrifuge use?
This is E. coli. The strain we use for this is specifically engineered to allow this to happen - this won't work with just any bacteria. In this case, the Bacteria's own beta-galactosidase gene is mutated in such a way that the enzyme subunit that is produced will only work if combined with the beta-galactosidase subunit encoded on the vector.
the centrifugation speed used in this lab isn't that important. It just has to be fast enough to pull things to the bottom of the tube. (I think we used the maximum speed in this case)