Zil Patel MCB 251 Unknown Vector Analysis PDF

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unknown vector analysis report for MCB 251 Lab...

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Zil Patel MCB 251 November 9th, 2017 Section T Analysis of an Unknown Vector Purpose: The purpose of this lab is to identify the gene from which an unknown insert is taken using the techniques we learned throughout the course of the semester. We learned how to use an unknown plasmid as a vector to clone the cat gene (BamHI digested) and the kan gene (BamHI digested). Also, we learned how to ligate genes into the vector DNA. Doing this procedure, we can successfully clone foreign pieces of DNA into vectors and successfully identify the inserts from the vectors. Procedure: In this lab, there will be two modes of analysis used of identify an insert piece using an unknown plasmid. PCR and plating will be used in order to identify the insert piece. We will need: 1. Vector DNA – the unknown plasmid isolated in week 4 and 5 2. Possible Insert DNA – cat gene (BamHI digested) and gfp genes 3. Restriction enzymes – HindIII and BamHI 4. LA Plates: LA and LA/Amp plates 5. Enzymes: DNA primers, Taq Polymerase, other reagents needed for PCR Using PCR, the insert gene cat will be amplified. We will then determine a target gene chosen from the insert gene – cat or kan. First, we need to do denaturation for PCR. The temperature will need to be 94 degrees Celsius in order to heat the target sequence an denature the DNA strands. Next, primer annealing takes place. The strands will be cooled to 50 degrees Celsius, allowing upstream and downstream primers that are specific to cat and gfp genes to anneal. Finally, the strands will undergo elongation, in which, the strands will be heated to 72 degrees Celsius to extend the primers with Taq polymerase. Taq polymerase is the best polymerase to use because it can withstand the high temperature. Thus, it can be used to determine whether the insert has been properly inserted into the vector or not. Adding these primers also allows for the strands to complementary base pair. Run the PCR thermocycler for 3 hours to denature the enzymes. After the 3 hours are over, put the products in the gel box for about an hour. Upon observation of the band. It can be determined that the insert was properly placed. After transformation and plating, we have a mixture of covalently closed circles and only a small fraction of that will be the desired DNA clone. In order to identify the clones, we need to introduce the DNA circles into the E. coli. Obtain the E coli colonies that contain the clone in them. This can be done by plating a mixture of the transformed bacterial cells on the LA/Ampicillin plate. Then, spread the bacterial cells on the LA plate with the ampicillin. The cells that do not take in the plasmid will die because both inserts have the ampicillin resistance

gene. If one observes that, there are bacteria growing there, then we know that we have the Kan gene. Ligation: We will make the restriction digest with BamHI to clone the insert into the vector. Insert genes are already digested with the enzyme. Because of this, the sticky ends with pair with each other and the plasmid will reform. Rejoining of the plasmids is more prevalent if the ends are closer rather than farther away. 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 three to one ratio of insert to vector concentration. In each Eppendorf tube, place: - 2.5 mL of vector DNA - 3 mL of insert DNA - 5 mL of buffer solution - 1 mL of T4 DNA ligase - 8 mL of water Plasmid Vector Insert #1 Explanation Convert mcl into ng/mcl 125 ng/mcl x 10 mcl digested 70 ng/mcl x 5 mcl digested vector pBLU / 30 mcl = 11.67 vector pBLU / 40 mcl = ng/cml 31.25 ng/cml 31.25ng/mcl x 10-9 g/ng = 11.67ng/mcl x 10-9 g/ng = Convert ng/mcl into g/mcl 3.125e-8 g/mcl 1.17e-8 g/mcl 5437 BP pBLU x 660 g/mol x 1000 BP pBLU x 660 g/mol x Calculate the mol/mcl for the BP = 3588420 g/mol BP = 660000 g/mol plasmid and insert Convert g/mcl to mol/mcl .1.17e^-8 g/mcl x 3.125e^-8 g/mcl x mol/660000 = 1.77e^-14 mol/3588420 = 8.71e^-15 mol/mcl mol/mcl Find the final amount of mcl .06 pmol x 10^-12 mol/pmol .02 pmol x 10^-12 mol/pmol x mcl/8.71e^-15 mol = 2.296 x mcl/1.77e^-14 mol = 3.390 to put in ligation mixture mcl Vector Pblue mcl Vector Pblue The total volume in each of the Eppendorf tubes should be 20 mL. For our control, we will have an uncut vector for which the ligase will be negative, a cut vector with a negative ligase, a cut vector with a positive ligase, and an insert or water with a positive ligase. Transformation: Obtain one tube of DH5a cells from the ice bucket. The tube 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. Next, add 4 mL from your uncut unknown purified plasmid to the tube containing 80 mL of chemically

competent cells labels “P”. Add no DNA to the tube labeled “NP”. Incubate on ice for 30 minutes. Then, place both of the tubes in the floating microfuge rack in the 42-degree Celsius water bath for 90 seconds. This is known as heat shock, which is used to destabilize the bacteria and encourage increased uptake of the recombinant vector. Next, place both tubes in the ice for 2 minutes. Add 800 mL of SOC broth to each of the microfuge tubes. Finally, place both tubes in your section’s microfuge rack to be taken to the shaker/incubator. The prep staff will take these and place them in the incubator. The cells will be grown at 37 degrees Celsius for one hour. Next, using the techniques for preparing a spread plate, inoculate a LB/ampicillin place with 100 mL from each of the 2 cultures. Prepare the plates one at a time to avoid the plates from drying out and getting contaminated. Make sure to label the plates with the culture you are inoculating with. After the plates are dry, place them upside down on the trays in the back to be incubated. All of the plates will be incubated at 37 degrees Celsius overnight and stored in the refrigerator until week 7. PCR: Obtain a pipette, a culture of cAMP #1 grown on the LA plate and two tubes – one containing a LB/Kan mixture and one containing an LB/Cat mixture. Use the pipette tip and probe half of a single colony onto the LA plate. Then, eject the pipette into the LA/Kan mixture. Then, incubate the tubes in 37 degrees Celsius for one week. These were the results that we got back: LA LA w/ Amp Cat Gene Growth Growth Kan Gene Growth Growth

LA w/ Kan No growth Growth

LA w/ Chl Growth No growth

Colony PCR: Obtain a pipette, a culture of pAMP #1 that was already grown on a LA plate, two tubes: one containing a LB/Kan mixture and on with the LB/Cat mixture. Use a swap to get one colony off of the demo plate for insert one and probe it on LA plate. Repeat the step for the LB/Cat mixture. Incubate the tubes for one week at 37 degrees Celsius. After the incubation, you can see the bacterial growth. The following table shows what growth determines which gene we would have: LB/Kan LB/Cat Kan Gene Growth No growth Cat gene No growth growth Conclusion: We hypothesized that insert #1 was Kan. Our PCR results did not come out as predicted, so we had to use the inoculation results. In the inoculation, there was growth in the LB/Kan Eppendorf tube, but not the LB/Cat tube, showing that the insert gene was Kan.

For our PCR plates, there was bacterial growth on the LA plates and one LA/Amp plate. Looking at the growth on the LA/Amp plate, we assumed it was contaminated because the bacterial growth did not look similar to the bacterial growth on the other plates. Next time, I would redo the streaking method and make sure that the swab we used has not been contaminated by surrounding air particles....