Day 1 of the workshop was great! Details of the same can be found here!
We were excited for Day -2, and that excitement didn’t go waste! 12-04-2016, i.e. day 2 of workshop we attended with a small glimmer of pre-knowledge! We performed 2 techniques: a) PCR and b) Restriction Digestion.
First session started with PCR.
PCR stands for Polymerase Chain Reaction. This technique is used to amplify or duplicate our strand of DNA and make it into multiple copies out of the same essentially the Control C-Control V mechanism of the cell. This technique is simple yet astounding on how it works. This technique needs 4 basic reagents,
- Template DNA
- Primer DNA (complementary to template DNA).
- Taq DNA polymerase
- dNTP’s (deoxynucleoside triphosphate)
PCR works by cycling between heat and cooling cycles. Where entire mastermix (with all necessary components) was rapidly heated to a desired temperature and then cooled back down. The exact temperature can be calculated using certain formulas based on the G-C ratio of the template DNA. The PCR cycle works in 3 stages, Denaturing, annealing and Extension. During denaturing the template DNA denatures into single stranded DNA, in the next step the primer which is complementary to template DNA binds to the template DNA. Using taq polymerase which is heat stable taq polymerase binds to the primer of the DNA and starts replicating the DNA in the 5” to 3” direction and starts amplifying the template DNA this is the first cycle and is repeated typically for 30-35 cycles. Until we get the desired copies of the original DNA affectionately called the “amplicon”.
By using a micropipette we added designated volumes of the reagents namely dNTPs, Distilled water, Buffer, and finally taq polymerase and 1 positive and 1 negative control was added in the negative control no template DNA was added in which no amplified DNA is visible which serves the purpose to provide a control for work. Then all the tubes were kept in PCR for 35 cycles alternating in between 77 celsius (hot phase) to 55 celsius (cold phase) In the meanwhile we prepared the agarose gel and when PCR cycle was done we added the 20 microliters of the sample and with a suitable Ethidium bromide dye we ran the Sample in the gel-electrophoresis box so that DNA will diffuse through the agarose gel and we can see well designated bands in the UV-Transilluminator.
The thought of amplifying DNA itself was so- exciting that the well designed process and its results in itself gave me so much to learn from. The applications of this techniques are immense right from forensics where the DNA can be amplified from small samples and the offender thrown behind the bars; upto amplifying new DNA used in recombinant DNA technology for multiplying new modified pieces of DNA opening new horizons for medicine and pharmacy ! Indeed the work-experience was great and the instructors made every concept clear as glass and guided our way through successful amplification.
In the second half, we headed for the second experiment of the day which was Digestion of vector with different restriction enzymes or simply Restriction Digestion.
Basically Restriction Digestion is a procedure used in molecular biology to prepare DNA for analysis or other processing. It is sometimes termed as DNA fragmentation also.
WHAT IS RESTRICTION DIGESTION ?
This enzymatic technique can be used for cleaving DNA molecules at specific sites, ensuring that all DNA fragments that contain a particular sequence have the same size; furthermore, each fragment that contains the desired sequence has the sequence located at exactly the same position within the fragment. The cleavage method makes use of an important class of DNA-cleaving enzymes isolated primarily from bacteria. These enzymes are called restriction endonucleases or restriction enzymes, and they are able to cleave DNA molecules at the positions at which particular short sequences of bases are present.
The class was divided into parts depending upon the Enzymes provided to us. We were provided with the plasmid used on 11th the first day of workshop which was pUC18. Groups were divided and were distributed with the Restriction Buffer, Enzyme and Autoclaved deionized water in set concentrations of 𝛍l. We added the reagents with the help of micropipettes which was quite a task in itself. The amount of concentration needed to transfer the reagents was a bit testing as we normally use pipettes upto 1 ml mark. The experience of handling the micropipettes was amazing.The enzymes added to the plasmid samples act as restriction enzymes which cut the DNA at its restriction sites.
Main four restriction digestion enzymes used were Pvu II, Bgl 1, BamH 1, EcoR1.
After addition of the reagents in specific order (plasmids,water,buffer and enzyme) the eppendorf tubes were put for incubation @ 37℃ for about 90 minutes. Meanwhile we prepared the tank buffer, agarose gel, buffer needed to load the overall mixture. After incubating the total set, it was then loaded into the Agarose gel which was prepared. The sample was added with the 6x gel loading dye and then transferred or loaded onto the agarose gel.
Agarose Gel-Electrophoresis was ran for about 30 minutes and then observed under UV light. It was done so that the DNA will diffuse and the bands can be observed. What we saw was the bands of DNA strands that diffused into the gels and the action of restriction enzyme on the plasmids. The fluorescent orange bands showed the migration of plasmid and cuts at certain base pairs.
Some of the images of the enzymes acting on the plasmid:
We learnt that Restriction Digestion is more suitable for analytical techniques such as Agarose Gel-Electrophoresis and Chromatography, as well as it is used in genetic fingerprint and RFLP analysis.
The experience we got performing the experiments which we only studied a little bit in theories so far, was an eye-opener about the vast applications of the techniques we perform in practicals and importance of them in the pharma industries.
Looking forward to today’s session as there is much more to gain from this precious workshop…
— Ajinkya Chitale
— Rajas Godbole