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The purpose of the gel might be to look at the DNA, to quantify it or to isolate a particular band. The DNA is visualized in the gel by addition of ethidium bromide. This binds strongly to DNA by intercalating between the bases and is fluorescent meaning that it absorbs invisible UV light and transmits the energy as visible orange light
PCR Lab
Purpose:
We are doing this experiment to teach you more about how to run a successful PCR. We also want to show you how one can determine if a DNA sample is bacterial by amplifying the 16S gene which amplifies at 1500 Kb. The 16S gene is unique to the domain Eubacteria. If you do not see the 16S gene amplified on the gel then it may be due to contamination from non Eubacteria DNA or from human error.
All 3 steps above are considered as one cycle; it takes about 25-40 cycles in order to amplify DNA template. The number of cycles depends on the amount of DNA available and how much PCR product you want to yield. To carry out the PCR we will use a thermal cycler that is programmed with a protocol that goes through all three steps of a cycle, for a total of 35 cycles. See PCR animation.
Since PCR is so sensitive we should always use a positive and negative control. The positive control should be a PCR mix with DNA known to work in amplification. The negative control should be a PCR mix without DNA. The experimental part should be your PCR mix with DNA.
micropipettes. Micropipettes come in different sizes and so do the pipette tips make sure you are using the correct pipette and tip for the amount you need to pick up. The p20 micro pipette is only accurate for picking up 2L - 20L A p200 micro pipette is only accurate for picking up 20L - 200L The p1000 micro pipette is only accurate for picking up 100L - 1000L VERY IMPORTANT when using the micropipettes be careful and avoid contaminating the micropipettes by slowly releasing the plunger. When using the micropipette you will need to stop pushing the plunger when you feel it stop the first time. The second stop will give you more than what you measured. Remember, if you are not sure about the last thing you did with your pipette tip then you should just get a clean one. Dirty pipette tips should all be autoclaved so they are to be discarded in the tin cans with red bags. Please balance the micro centrifuge machine by placing another tube across from your tube both tubes should have approximately the same amount of liquid, remember you are balancing. You may use the balance tubes or another students micro centrifuge tube. Make sure you know where you place your tube or you will not know which tube is which. The best thing to do is to label the micro centrifuge tubes (LABEL EVERYTHING). Or else you will have to start over. If you have never used a micropipette practice with autoclaved DI water ~Procedure~
PCR protocol
The next step is adding all the PCR reagents together into one sterile micro centrifuge tube, labeled Master Mix. Since the amounts necessary for a single 50L PCR reaction are difficult to measure each lab will make 1 master mix. Below is the breakdown of what amount is measured into each micro centrifuge tube per number of reactions.
# of reactions
Promega Master mix (L)
25
625
1494 R primer (L) 12.5 530 F primer (L) 12.5 Nuclease free H20 (L) 612 Total (L) The amount of reagent per 50L reaction is as follows these numbers are shown so that you can see what your own reaction should have. Do not try to measure 0.26L you wont be able to see it and we dont have a micropipette that measures that small amount. # of reactions 1 Promega Master mix (L) 25 1494 R primer (L) 0.5 530 F primer (L) 0.5 Nuclease free H20 (L) 19 Total (L) 45 If you add up the total for 1 reaction youll notice it is missing 5L of the cell suspension that you will add to the PCR tube NOT the master mix. Making the Master Mix (TA): 1. Turn the dial of the micropipette so that you are measuring the proper amount of PCR reagent for your group. 2. All of the PCR reagents must be thoroughly mixed just prior to measuring - flicking the tube of the reagent you need to measure does this. Make sure all the liquid has uniform turbidity. 3. DO NOT vortex the PCR reagents, this will destroy them. 4. Put the proper micropipette tip on the micropipette. 5. Push the plunger down to the first stop 6. Be sure ONLY the pipette tip touches the inside of the tube and slowly release the plunger and be sure you picked up the liquid you are measuring. Avoid contamination. 7. Put your liquid into the proper tube by pushing the plunger to ONLY the first stop. 8. PCR reagents go into the Master Mix micro centrifuge tube. The Student: 9. When done with the Master Mix, mix it well, and pipette 45L of it and add it to your own PCR tube. Close your tube and place in ice. 10. When the class is done with PCR tubes they must all be mixed by flicking just prior to loading into the thermal cycler when it has reached a temperature of 95 C BE CAREFUL the cycler is HOT and will burn your skin if the plate is touched just insert the tubes and lock the cycler.
protection must be used as well. PCR tubes with product have been stored at -20C; they need to be thawed in the fridge at 4C or in ice. This should be quick because you only have 50L in these PCR tubes. Your TA should also thaw the ladder, which is a DNA marker that will be used to show you where the 1500 Kb is located on the finished gel. The ladder is mixed with nuclease free H20 and some 6X dye. TAs will load the ladder and the positive and negative controls. The electrophoresis gel is in a buffer solution that ensures the DNA will be negatively charged. The gel also has 32 wells in it that are formed with combs inserted into the gel before it has hardened. You will pipette your PCR product + dye into these wells. When doing this you need to be careful not to break the gel, which has a consistency of gelatin. To avoid breaking the gel, steady your hand and place the pipette tip in the buffer solution just above the well you chose. DO NOT TOUCH the gel you dont need to touch it with the pipette tip. Just slowly push the plunger to the first stop and your solution will fall right into the well. If this is done successful you have loaded a well. If you break a well let your TA know. The reason the product will fall is due to the dye you added to the PCR product the dye makes the product heavy. This dye is also used so you are able to see what wells have product and when the gel needs to be stopped. If a gel is never stopped the product runs right off the gel and into the buffer. To stop a gel all you need to do is turn off the electricity. ~Procedure~ 1. Take 12 L of your thawed PCR product in a p20 micropippete. 2. Bring micropippete to gel, and carefully place tip near one unused well for dispensing. 3. Release product slowly into gel, allowing it to fall into the lower well. 4. The green dye has a reagent that allows the product to be heavier than the buffer it is in therefore allowing it to fall into well. Once everyone has put their PCR products into the wells the electrodes need to be checked and the machine needs to be turned on at 150V because it is such a large gel. Why check the electrodes? Because you want your gel to run from where you loaded the wells to the bottom of the gel, if it goes the wrong way you will run your product into the buffer. You need the negative electrode on the loaded wells side and a positive electrode on side without wells; that way your product, which is negatively, charged moves towards the positive end. Running a gel takes about 40 60 minutes. When the gel is done a picture will be taken and you can check to see if you properly ran a PCR and gel and see the proof of the 16S gene amplified at 1500 Kb.
Gel Outcomes:
* A positive result for bacteria will have the 16S Gene deposited at 1500 kB, such as the cartoon rendition in Well 2. Acknowledgements I would like to thank David Chang, Shyama Malwane, Linh Nguyen, and Dr. Eduardo Robleto for their expertise and support. References White, Bruce A., ed. PCR Cloning Protocols: From Molecular Cloning to Genetic Engineering. Methods in Molecular Biology, Volume 67. Totowa: Humana Press Inc 1997. Berg, J.M., Stryer, L., and Tymoczko, J.L. Biochemistry 5th ed. New York: W. H. Freeman and Company, 2002.
Revised by N. Fester on November 7, 2005