PAREKH Unknown Insert Lab Report

.docx

School

University of Illinois, Urbana Champaign *

*We aren’t endorsed by this school

Course

251

Subject

Biology

Date

May 1, 2024

Type

docx

Pages

Uploaded by DeaconHyenaMaster878 on coursehero.com

Rhea Parekh Unknown Insert Lab Report Purpose Adding vectors to a plasmid and performing transformation is useful in science since it inserts foreign DNA to a plasmid. These plasmids can be replicated and be used to learn about the characteristics of a gene and study the proteins expressed. The objective of this laboratory experiment was to test combine the skills learned throughout the semester to identify an unknown plasmid insert. The various microbiological procedures that are going to be used in this protocol are Qiagen plasmid purification, spread plating, restriction digest, PCR, and gel electrophoresis. We injected unknown vector #2 into pBLU plasmid. The unknown vector could be cat or kan. The cat gene contains resistance to the antibiotic chloramphenicol and the kan gene has resistance to the antibiotic kanamycin. Both inserts are of different lengths. To identify the unknown insert, we experimented using two modes of analysis: transformed cell growth on LA + antibiotic plates (Plating) and PCR. Procedure The laboratory procedure was structured into two distinct steps to accommodate two modes of analysis. The initial step involved Plating, encompassing restriction digestion, ligation, and subsequent transformation. Following this, the plasmid carrying the unknown insert underwent cultivation on petri dishes. Upon successful bacterial cell growth with the unidentified insert, we proceeded to miniprep, followed by plasmid purification. Post plasmid purification, a clean plasmid was obtained for the second step involving PCR. Following the completion of PCR, gel electrophoresis was conducted on the amplified DNA fragments, and the results were subsequently observed. I. FIRST MODE OF ANALYSIS- PLATING Restriction digestion The initial step in determining unknown plasmid is restriction digestion. Restriction digestion is a molecular biology technique that involves the cleavage of DNA at specific recognition sites by enzymes known as restriction endonucleases. These enzymes recognize specific DNA sequences, typically palindromic sequences, and cut the DNA at or near those sites. We employed specialized proteins known as restriction enzymes to precisely cleave DNA at specific recognition sequences. We used BamHI restriction enzymes. When applied to DNA, BamHI accurately cleaves it at predefined sites, yielding DNA fragments with "sticky ends" that possess complementary sequences.

Rhea Parekh This precise cleavage is a cornerstone of cloning, as it ensures the accuracy of gene insertion. To execute this process, we followed a structured set of steps. - Restriction enzyme BamHI was selected to cut pBLU plasmid. We obtained a microfuge tube and labeled it as “P” for pBLU restriction digestion. A reaction mixture was then set up which included 5 ul of pBLU plasmid, 1 ul of BamHI, 12 ul of H20 and 2 ul of buffer, preparing the total volume of 20ul. The tube was gently inverted to mix all the contents together. Restriction enzymes and ligases have opposing functions. We incubated it for an hour at 37 degrees. - This means that as long as active restriction enzymes are present, the ligase activity is going to be compromised. Thus, we heated the reaction mixture in a thermocycler for 20 min at 65 degrees celsius. After heat shock, the tube was incubated overnight at 37 degrees celsius. - The insert was already digested with BamHI so restriction digestion was not necessary for the insert. Ligation The ligation process follows DNA cleavage by a restriction enzyme, aiming to rejoin DNA fragments and create a recombinant DNA molecule that incorporates our gene of interest and vector DNA. T4 DNA ligase is employed to seal any nicks or gaps in the DNA strands by catalyzing the formation of phosphodiester bonds. Typically, a 3:1 ratio of vector to insert is used in ligation reactions. To set up this reaction, we started by labeling a new microcentrifuge tube as 'T' for T4 DNA ligase and calculated the required volumes based on the 3:1 ratio and specific DNA concentrations. Calculations - concentration and volume of pBLU and insert pBLU- we first need to find the final concentration of pBLU plasmid through m1v1= m2v2. The initial concentration of pBLU was 50 ul/ml and initial volume was 5 ul. The final volume of pBLU was 20ul. With the equation m1v1= m2v2, (50)(5) = (x)(20). We found out that the final concentration of pBLU plasmid was 12.5 ul/ml. We also need to calculate molar mass of pBLU which can be found by multiplying the size of the plasmid( in bp) by the average molecular weight of a DNA base pair(650 g/mol). Molar mass of the plasmid = Average molecular weight of DNA X size of the plasmid. Average molecular weight of pBLU is 5437 5437 bp X 660 g/ 1bp = 3588420 g Finding volume- From digestion step, we calculated the new concentration of pBLU in ng/ul and we now need to convert that into pmol/ul because ultimately we need 0.05 pmol of vector DNA and .15 pmol of insert DNA. 12.5 ng/ 1 ul X 10^-9 g/ 1 ng X 1 mol/ 3588420 g X 10^12 pmol/ 1 mol = 0.003484272 pmol/ ul = 3.48343e-3 pmol/ ul - Using 0.05 pmol vector DNA

Rhea Parekh 0.05 pmol X 1 ul/ 3.48e-3 pmol = 14.37 ul Insert 2 The size of the Kan gene is 1478 bp and the cat gene is 1015 bp . We can take the average of both genes to find the molar mass since we don’t know which one we have. 1246.5 bp X 660 g/bp = 822690 g The concentrations for the unknown gene inserts were already given. For unknown 1-> 110 ng ul We then converted this concentration into pmol/ul 110 ng/ 1 ul X 10^-9 g/ 1ng X 1 mol/ 822690g X 10^ 12 pmol/ 1 mol = 0.134 pmol/ ul We then used the concentration calculated and 0.15 pmol insert DNA to calculate the volume. 0.15 pmol X 1 ul/ 0.134 pmol = 1.122 ul Plasmid (pBLU) Insert Molar mass (g) 3488420 822690 Concentration( pmol/ul) 3.48343e-3 0.134 Volume (ul) 14.35 1.122 This table shows the concentration and volume calculated from the concentration of plasmid and insert. Once all these calculations were done, we obtained a microfuge tube and labeled it “L” for ligation. The insert was added to the plasmid. Reagents volumes ( ul) Vector DNA 14.4 Insert 2 1.2 Ligase buffer 2 Ligase 1 H20 1.4 Total 20

Rhea Parekh This table shows how much volume has been added for each reagent to get the total volume of 20 ul. In the lab, Once all the reagents were added, the microfuge was gently inverted and then incubated overnight at 37 degrees. Transformation Transformation, a pivotal process in molecular biology, involves introducing foreign DNA, like a plasmid housing a gene of interest, into a host organism, typically a bacterial cell. The initiation of transformation was carried out by preparing a microfuge labeled as "P" and obtaining competent cells. In this process, 80 μl of competent cells were combined with 4 μl of the ligated product after incubation, followed by a 30-minute incubation on ice. Subsequently, the tube underwent a 90-second placement in a floating microfuge rack and then returned to ice for an additional 2 minutes. After adding 800 μl of SOC broth, the tube was incubated in a shaker/incubator at 37 degrees Celsius for 1 hour. Plating the bacterial culture Cultural plates, including LA, LA/AMP/X-gal, LA/Chl, and LA/Kan plates, were then acquired, with those containing multiple antibiotics labeled accordingly. Using a Bunsen burner, a glass spreader was sterilized, and 100 μl of cells were gently and evenly spread on each plate while rotating it swiftly. Once dry, the plates were inverted on trays for overnight incubation at 37 degrees Celsius and subsequently stored in the refrigerator. This process was repeated for each set of cells dispensed onto the plates. II. Second mode of analysis- PCR Upon observing successful growth of our culture containing an unknown gene on the anticipated plates, we identified our unknown inserts cat. To ascertain the presence of the cat gene, we expected growth on LA/Chl plates, and indeed, growth was observed. The process of transferring cells from a solid medium (like an agar plate) to a liquid medium (broth) is commonly referred to as "inoculation" or "subculturing." Inoculation involves transferring a small amount of cells, typically using a sterile inoculating loop or swab, from a solid culture to a liquid culture.To proceed with the inoculation,, we initiated the process by inoculating colony of bacteria from the LA/Chl plate, which contained the desired plasmid with the cat gene. This colony was introduced into a small volume of liquid culture medium supplemented with chloramphenicol. The inoculated culture was then incubated overnight at 37 degrees Celsius. This step is

Rhea Parekh crucial for allowing the bacteria to grow and maintain the plasmid with the cat gene under selection pressure. Plasmid Purification Minipreep is purifying the plasmid where you let the cells grow on the broth and this is used to do plasmid purification. The process of plasmid purification entails isolating and purifying plasmid DNA from bacterial cells. To initiate the procedure, a liquid culture from the previous week, where bacteria was allowed to grow, was obtained. Five microfuge tubes, labeled as "1-5," were prepared, and 750 µL of bacterial culture was added to each microfuge using a p1000 pipette. Following a 60-second centrifugation, the supernatant was discarded. Subsequently, 250 µL of P1 was added to the microfuge labeled as "1," and the contents were thoroughly mixed using a micropipette. This mixture was then transferred to the microfuge labeled as "2," and this process was repeated until the last microfuge labeled as "5." To the final tube labeled as "5," 250 µL of P2 was added, inverted 4-6 times, and allowed to sit for 4 minutes. Following this, 350 µL of buffer N3 was introduced to the cell lysate, and the tube was inverted 4-6 times before being centrifuged for 10 minutes. After the centrifugation, 750 µL of the supernatant from the tube was added to a Qiagen column and centrifuged for 60 seconds. The effluent was discarded, and 500 µL of buffer PB was added, followed by another 60-second centrifugation and effluent disposal. Finally, a 30-second dry spin was performed, and the spin column was transferred from the trap to a fresh sterile Eppendorf tube in a microcentrifuge for an additional 30 seconds. We then had a clean plasmid suspended in EB. Quantification of Plasmid DNA After plasmid isolation, we used a spectrophotometer called nanodrop to measure DNA yield and purity. The concentration of DNA was 71.350 ng/ul. Our value of A260/A280 was 1.799 which is close enough to 1.8 for us to say that our sample was relatively pure and not majorly contaminated. A value less than 1.8 indicates contamination. PCR Polymerase Chain Reaction (PCR) is a foundational molecular biology technique employed for the targeted amplification of specific DNA segments. In our procedure, we initiated the PCR setup by acquiring the Paq5000 Master Mix (MM), Plasmid DNA, Upstream Primer, and Downstream Primer from the designated area on the laboratory bench. We mixed 12.5 ul of PCR MasterMix, 2.5 ul of Template DNA, and 5 ul each of upstream and downstream primers. Following gentle pipetting to ensure thorough mixing and centrifugation, a 25 μl aliquot of this mixture was carefully transferred to a PCR tube. Subsequently, the PCR tube was placed into the PCR thermocycler, where the amplification reaction ran for a duration of 2.5 hours. Once the PCR cycle was completed, the product was run out on gel electrophoresis.

Rhea Parekh - 5 ul of loading dye with 10 ul of the PCR mixture was mixed. All 15 ul of the mixture was loaded into the gel wall 6. The agarose gel ran for 60 minutes at 100 V. - The results were then observed and analyzed Results and data I. FIRST MODE OF ANALYSIS This image shows bacterial cells plated on LA plates without any additional agents. Without any additional selective pressure, all transformed cells, regardless of the presence or absence of the plasmid, would be able to form colonies on LA plates.

Your preview ends here

Eager to read complete document? Join bartleby learn and gain access to the full version