PAREKH Unknown Insert Lab Report
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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.
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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
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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.
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as
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common-assessment-delivery/start/4797160920?action=onresume&submissionld%-468979669
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Lab Report 6 worksheets 314 F22 .DOCX
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Grades for Arysta Visser: 23 x M Uh-oh! There's a problem w X b The restriction EcoRI cleaves X + Untitled spreadsheet - Goog X
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18. The restriction EcoRI cleaves double-stranded DNA at the sequence 5'-GAATTC-3', the
restriction enzyme HindIII cleaves at the sequence 5'-AAGCTT-3', and the restriction enzyme
BamHI cleaves at 5'GGATCC-3. An 805 bp circular plasmid is digested with each enzyme
individually and then in combination, and the resulting fragment sizes are determined by
means of electrophoresis. The results are as follows:
1
Restriction Enzyme(s)
EcoRI
BamHI
HindIII
EcoRI and BamHI
EcoRI and HindIII
BamHI and HindIII
3
Practice
====•=•€ EX
Fragment lengths (base pairs)
430 bp, 375 bp
470 bp, 335 bp
Lab Report 6…
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The objective is to interpret the results of an RNA-Seq analysis to identify differentially expressed genes in breast cancer using figure 1. The data provided includes gene symbols, chromosome location, start and end points, strand, fold change, log2 fold change, p-value, and false discovery rate (FDR). The RNA-Seq analysis has identified several genes that are differentially expressed in breast cancer. These genes are located on various chromosomes and have varying levels of fold change, indicating the degree to which their expression levels differ between normal and cancerous cells. The gene with the highest fold change is EYA4, located on chromosome 6, with a fold change of 3604.4176. This indicates that the expression of this gene is over 3600 times higher in cancer cells compared to normal cells. The log2 fold change is 11.81555, which is a measure of the magnitude of the difference in gene expression. The…
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Name
Section
date sheet
MAPPING PRACTICE #1
Below is a restriction map for the plasmid PGEN 101 (total length = 20 Kb). Using this map as a
guide, give the number of restriction fraqments along with their associated lengths that would result
from digesting PGEN 101 with the restriction enzymes EcoRI, BamHI and a combination of ECORI and
BamHI.
BamHI
3.2 Kb
1.7 Kb
EcoRI
BamHI
PGEN 101
8.7 Kb
5.5 Kb
.9 Kb
EcoRI ECORI
DIGESTION PERFORMED
SIZES OF FRAGMENTS OBTAINED
10.4 kb , 0.9kb, 8.7 Kb
EcoRI
3.2 Kb, 16. 8kb
BamHI
EcoRI + BamHI
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3. Recombinant protein is produced by a genetically engineered strain of Escherichia coli during cell
growth. The recombinant protein can be considered a product of cell culture even though it is not
secreted from the cells; it is synthesized in addition to normal E. coli biomass. Ammonia is used as
the nitrogen source for aerobic respiration of glucose.
The recombinant protein has an overall formula of CH1.5500.31N..25. The yield of biomass (excluding
recombinant protein) from glucose is measured as o.48 g/g; the yield of recombinant protein from
glucose is about 20% of that for cells.
How much ammonia is required?
What is the oxygen demand?
If the biomass yield remains at 0.48 g /g, how much different are the ammonia and oxygen
requirements for wild-type E. coli that is unable to synthesize recombinant protein?
а.
b.
с.
24°C
08:39
Berawan
27/04/2022
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question on plasmid minipreps and restriction digestion
How does this procedure allow you to purify the plasmid DNA away from the chromosomal DNA?
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Plate
LB-Plasmid
LB + Plasmid
LB/amp - Plasmid
Yes
Yes
No
+ LB
LB/amp + Plasmid Yes
LB
Prediction
Reeson
Plate only contains Yes
food/bacteria
Plate only contains Yes
food/bacteria
Bacteria will be
killed by ampicillin
Bacteria has
ampicillin
resistance
LB
Click on pic to zoom Click on pic to zoom
-LB
Observed Result
No
Yes
chlamp
x
Click on pic to zoom
+ LB/AMP
Blamp
Click on pic to zoom
- LB/AMP
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I need help writting a biological "title" for this lab report. I performed 5 labs which are PCR purification, Rapid ligation, and Transformation, Blue/White screening and observation of pGreen transformation, Plasmid Extraction and Restriction Enzyme Digestion, Soil DNA extraction and gel electrophoresis, and Bioinformatics Analysis of 16s rRNA genes.
Note; I attached one page of my abstract and one page of the introduction
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Incorrect
1
A
Help Center?
It can sequence a whole genome in one huge strand
Each nucleotide has a different marker attached
Question 11
LO62 Explain how SANGER sequencing works
Order the steps that are needed for gene sequencing through Sanger technology:
Amplify DNA
Cut DNA into small pieces
Add DNA to be sequenced + DNA
polymerase + dNTPs + ddNTPs into
different test tubes
Run resulting sequences through gel
electrophoresis
3
111
0/1 pts
0 / 1 pts
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Pipetting
1. Explain why a micropipettor is an important instrument in biochemistry labs.
2. Describe the basic components (and function) of a micropipettor.
Bacterial Techniques
1. Define the following.
(a) Serial Dilution
(b) Streak Plating
(c) Spread Plating
Transformation
1. Define bacterial transformation and explain why it is an important method in
biochemistry labs.
2. Describe (figure, narrative) a plasmid and describe the basic components of a
plasmid.
3. What role does CaCl2 play in bacterial transformation?
4. What role does heat shock play in bacterial transformation?
Plasmid Isolation
1. Describe the roles the following play in plasmid purification.
(a) Lysis Buffer
(b) Neutralization Buffer
(c) Elution Buffer
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Section
Name
MAPPING PRACTICE #4
Plasmid pBR 607 is a 2.6 Kb plasmid containing Ampicillin and Tetracycline resistance markers, an
origin of replication, and unique restriction sites for the restriction enzymes EcoRI, BamHI, and Pstl.
Given the restriction map for pBR 607 for the enzymes EcoRI, BamHI, and Pstl, show on the gel
diagram, where the approximate positions of the restriction fragments generated from the restriction
digests would be located after carrying out electrophoresis.
BamHI
0.2 Kb
pBR 607
ECORI
1.94 Kb
0.46 Kb
Pstl
Size
EcoRI
EcoRI
EcoRI +
Standards
BamHI +
Pstl
BamHI
Pstl
4.0 Kb
2.2 Kb
2.0 Kb
0.5 Kb
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I need help with determining size fragment
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- Please answer fast After the transfer of the F plasmid is complete multiple choice 3 the F+ cell becomes F-. the F- cell becomes F+. F+ cell becomes antibiotic sensitive. the F+ cell expresses an R factor. the F- cell becomes HfRarrow_forwardActivity 2. You COMPLETE Me! Objective: Identify the methods used for inserting plasmids into the host cells. What you need: pen and paper What to do: On a separate sheet of paper, copy and complete the key points of the methods used to introduce plasmids into the host cells. Choose your answers from the box gene gun biolistics electric shock mammalian cells plasmid insertion by Heat Shock Treatment increase the pore sizes of their plasma membranes (2) competent plasmid DNA Heat Shock electroporation There are three methods on introducing plasmids into the host cells namely: (1)_ (3) Biolistics uses a (4)_ to fire DNA-coated pellets on plant tissues. Plasmid insertion by Heat Shock Treatment is a process wherein target cells undergo a The pretreatment is said to make the cells (6). pretreatment procedure to (5) the introduction of the (7)_ plasmid at about 4°C for about 30 minutes and the plasmids concentrate near the cells during After the pretreatment, the cells are incubated with…arrow_forwardWrite TRUE or FALSE. If false, write the word/s that make(s) the statement incorrect. 1.The primosome complex includes primase, helicase and gyrase. 2.In plasmid DNA biotechnology, plasmid inserted with a “correct" DNA sequence is transferred to bacteria for rapid.arrow_forward
- Please help me answer topic is about making a recombinant DNA modelarrow_forwardComplete the sentences. Each term may be used more than once. In a circular bacterial chromosome, the structure of DNA is a If DNA is twisted in the Overwinding results in If DNA is twisted in the Underwinding results in One effect of double helix. direction, it becomes overwound. supercoiling. direction, it becomes underwound. supercoiling. double helix. Answer Bank positive negative right-handed left-handed supercoiling in bacterial chromosomes is to promote separation of the two strands of DNA in thearrow_forwardAnswer the ff. questions: 1. At eukaryotic origins of replication, helicase cannot be activated until the polymerase is also positioned on the DNA. Explain what would happen if the helicase became active in the absence of DNA polymerase. 2. RNA synthesis is much less accurate than DNA synthesis. Why does this not harm the cell? 3. What is the role of folate in the synthesis of nitrogenous bases of purines?arrow_forward
- ☆回 w Insert Format Tools Add-ons Help Last edit was yesterday at 8:08 AM TURN 100% Normal text Arial 11 BIUA E- 1E E E - E EX 2. 3 . 5. C Directions: Describe the process of DNA replication in the space below. Specifically reference the pictures in your answer. Your response will undergo a plagiarism check. Be sure to write your response in your own words. Be sure to reference and thoroughly explain: The relationship and importance of DNA replication and the cell cycle. The conservation of genetic information during replication. (Is it conservative? Semi-conservation? Dispersive? What does this mean?) The specific enzymes used in each step and indicate whether each of the enzymes is a synthesis reaction or a digestion reaction. Justify your answer through explanation. Identify the parameters and limitations of the enzymes working effectively. (What factors would limit or interfere with DNA replication?)arrow_forwardComplete the sentences about unusual DNA structures. is a DNA repeat that does not have a complementary sequence in the same strand. some secondary structures. Any sequence, such as a palindrome, that can be rotated 180° horizontally and 180° vertically such that it superimposes a sequence repeat on another sequence is In refers to self-complementarity within a strand. This sequence may contribute to the formation of A four-stranded, right-handed helix formed by a DNA segment containing a high proportion of guanine residues is known as The sequence An example of an inverted repeat that occurs within a single DNA strand is -GTGAG... .CTCAC- -CACTC. .GAGTG- interstrand hydrogen bonds break and intrastrand hydrogen bonds form. .. is which has the potential to form can form when a polynucleotide strand forms major groove of a homopurine-homopyrimidine duplex that contains a mirror repeat. A single DNA strand with the sequence - ATATG with functional groups in the CATAT- can form a…arrow_forwardU have the plasmid pUC18/19, which is a circular plasmid that consists of 2686 bp. What would the number of and length of the fragments be if you cut the plasmid with the following restriction enzymes or combination of enzymes? Give a schematic representation of the digestions. PscI & GsuI ______________________________________________________________ ScaI, PdmI & BsaXI ______________________________________________________________ ScaI, SspI & EheI ______________________________________________________________arrow_forward
- Restriction mapping of the delta chromosome I need help with question two pleasearrow_forwardcommon-assessment-delivery/start/4797160920?action=onresume&submissionld%-468979669 English 9 Student D. S ddddddddddddddd... Type of Genetics Cr. S TEWWG Chapter DNA Protein Synthesis Test Chromosome Free Nucleotides DNA Polymerase Leading Strand Original- (Template DNA) Helicase Lagging Strand Replication Fork Adenine Thymine Guanine Cytosine DNA Polymerase Original (Template) DNA Strand 1. This diagram is showing the process of 2. 1f the original template strand reads: TTACGCAGT, DNA polymerase would add nucleotides in the order: 4 5 6 7 8. 9 10arrow_forwardA amp PBR322 4301 fot B Clear Zones Figure 2 The postgraduate student, Demika, inserted her gene of interest into the plasmid, pBR322, before transformation into the competent host cell using heat shock method. After that she cultured the cells on the Ampicillin agar plate before replica plating the colonies onto another Ampicillin (A) and Tetracycline (B) agar plates shown in Figure 2. (1) Referring to the vector pBR322 in Figure 2, which recognition site was cleaved to insert the gene of interest? Based on the observation above, can you identify which colonies are carrying positive mcombinants of BR322? Explain your selection.arrow_forward
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