ZPExp Design WK 7 - Grade: A PDF

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Experiment design for week 7 procedure ...

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Zil Patel MCB 251 Week 7 Experimental Design #3: Identifying an Unknown Plasmid Purpose: The purpose of this lab is to learn to use an unknown plasmid as a vector in order to clone 2 genes: cat and kan. We also learn how to ligate genes into the vector DNA. Through this procedure, we can successfully clone foreign pieces of DNA into vectors and successfully identify the inserts from the vectors. Experimental Plan and Description: Within this lab, we will have two modes of analysis in order to identify the insert piece using an unknown plasmid. These two modes will be plating and PCR. In order to accomplish this, we will need the following materials:

1. Vector DNA – unknown plasmid isolated in week 4 and 5 2. Possible insert DNA: cat gene (BamHI digested) and kan (neo) gene 3. Restriction Enzymes – HindIII and BamHI 4. LA plates: LA and LA/Ampicillin 5. Enzymes: DNA Primers, Taq Polymerase, and other reagents required for PCR

By using PCR, the insert gene cat will be amplified. After that, we will use a target genes picked from the insert gene, which can be either cat or kan. For our first step of PCR, the temperature will be at 94 degrees Celsius to heat the target sequence and denature the strands of DNA. Next, in order to anneal the primer, the DNA strands will have to be cooled to 50 degrees Celsius, allowing the upstream and downstream primers specific to the cat and kan genes to anneal. Finally, the strands will undergo either extension or elongation because they will be heated to 72 degrees Celsius to extend the primers with Taq polymerase. Taq polymerase is perfect because it can withstand high temperatures and can be used to determine whether the insert has been inserted into the vector properly. Adding the primers also allows the strands to complementary base pair. After that, we will run the PCR thermocycler to denature the enzymes for about 3 hours. Afterwards, we can place the products back into the gel box for 60 minutes.

After observing the band, we can determine whether the insert was properly inserted or not. After transformation and plating has occurred, we will have a mixture or covalently closed circles and a small fraction of the desired clone. In order to identify the clones, we will introduce the DNA circles into E. coli. We will have to obtain the E. coli colonies that contain the clone in them. We can do this by plating a mixture of transformed bacterial cells on the LA/Ampicillin plate. Then, we will spread the bacterial cells on to the LA plate with ampicillin. The cells that did not take in the plasmid will die out as both inserts will have the ampicillin resistant gene (we assume). If one observes that there are bacteria growing there, then you know you have the cat gene. Ligation: Restriction digest will be made with BamHI to clone the insert into the vector. Insert genes are already digested with the enzyme, so the sticky ends pair with each other and the plasmids reform. Because the ends of the DNA are so close, rejoining is very frequent. A high ratio of foreign fragments to plasmid DNA must be used to increase the frequency of the foreign pieces that join with the plasmid ends. For our standard cloning, we will use a 3:1 ratio of insert to vector concentration. We will put each of the following into an Eppendorf Tube. 1. 2.5 mL vector DNA 2. 3.1 mL insert DNA

Total Volume: 20 mL

3. 5 μ 1 of buffer 4. 1

μ 1 of T4 DNA ligase

5. 8

μ 1 of water

B 10 μ 1 pblu 7 μ 1 water 3 BamHI TOTAL: 20 μ 1

Be 10 μ 1 pblu 6 μ 1 water 2 μ 1 BamHI & 2 HindIII TOTAL: 20 μ 1

Our control will be an uncut vector with a negative ligase, a cut vector with a negative ligase, a cut vector with a positive ligase, and an insert or water with a positive ligase. By doing so, we will be checking for viability of the competent cells, as well as, verifying the antibiotic resistance of the plasmid. We will then insert it in water to indicate whether there has been any contamination of the intact plasmid in the ligation or transformation reagents.

Transformation:

Obtain one tube of DH5a cells from the ice bucket. This tub contains 170 mL of competent cells. Label 2 sterile microfuge tubes “P” for plasmid and “NP” for no plasmid. Aliquot 80 mL of competent cells into each of these labeled microfuge tubes. Add 4 mL from the uncut unknown plasmid to the tube containing of 80 mL of chemically competent cells labeled “P.” Make sure to add none to “NP”. Incubate on ice for a span of 30 minutes. Then, place both tubes in the floating microfuge rack in the 42 tubes in ice for 2 minutes. Add 800

°C

water bath for 90 seconds. Next, place both

μ l of SOC broth to each of the microfuge tubes. Lastly,

place both tubes in your sections microfuge rack to be taken to the shaker/incubator. Prep staff will place these in the incubator to grow the cells at 37

° C for one hour.

Next, using the technique to prepare a spread plate, inoculate a LB/ampicillin plate with 100 μ l from each of these 2 cultures. (Transformation on an uncut plasmid only indicates growth on ampicillin). Prepare the spread plates one at a time so the cultures do not dry out. Make sure to label the plates with the culture you are inoculating with. When the tubes are finally dry, place them upside down on the tray at the back of the lab so they can be incubated. All plates are to be incubated overnight at 37

° C and then stored in the refrigerator until week 7....