1107L-GI

Kpnl 4. Plasmid Z has a size of 7 kb, and the map shows Kpnl (K) and Pstl (P) cut sites relative to each other. This plasmid was digested with three different restriction enzymes 2000 bp 3500 bp Kpnl (K), Pstl (P) and Bgll (B) either alone or in combination and the samples run on an agarose gel as shown below. Where does Bgll (B) cut this plasmid ? Does the plasmid have one recognition site or two for Bg|l? Describe the Bgll cut site in this plasmid relative to the Kpnl cut Plasmid Z -7 kb Pstl site. How many bases to the left or right of the Kpnl cut site would you observe the Bgll cut site. Explain briefly. 1500 bp Pstl Ladder Kpni Psti K/P Bgl K/B KPB 7000 bp 7000 bp 5600 bp 5500 bp 4900 bp 3500 bp 2000 bp 1500 bp 1500 bp 1500 bp 1400 bp 600 bp %3D

Restriction digestion and Gel electrophoresis: A single strand of a double-stranded DNA sequence is shown below. Draw a complementary DNA strand and show the restriction digestion pattern of the double-stranded DNA with BamHI and Pst1. Show the separation pattern of the undigested and the digested DNA on your agarose gel. Label the gel appropriately. 5’ – CGAGCATTTGGATCCTGTGCAATCTGCAGTGCGAT – 3’

Restriction mapping sample question You have a 5.3 kb PstI fragment cloned into the PstI site of the vector pUC19, which is 2.7 kb in size. This vector has unique sites for the following enzymes in a multiple cloning site: PstI, HincII, Xbal, BamHI, SmaI, EcoRI A restriction map of the 5.3 kb insert is prepared. The recombinant plasmid is digested with the enzymes listed above in single digests, and then several combinations of enzymes are tested in double digests. The following bands are observed when the digests are run on a gel: Enzyme(s) used PstI ECORI HincII Band sizes observed (kb) 5.3, 2.7 5.4, 2.6 4.5, 3.5 6.7, 1.3 | 4.0 (high intensity band) 3.9, 3.7, 0.4 4.0, 3.5, 0.5 3.5, 2.6, 1.9 3.7, 3.6, 0.4, 0.3 3.7, 2.2, 1.7, 0.4 3.7, 3.0, 0.9, 0.4 3.9, 3.5, 0.4, 0.2 Smal Xbal ВатHI HinclI + Xbal HincII + ECORI XbaI + BamHI ECORI + BamHI Smal + BamHI HincII + BamHI Use the data above to construct a map of the cloned insert. Note that fragments smaller than 100 bp will not usually be…

PCHEM4321. An agarose gel electrophoresis pattern of the plasmid PSPM4321 digestion (restriction) is shown below. Draw a restriction map of a plasmid with the appropriate restriction sites based on the data given below. Hindlll Hindll BamHI +BamHI Figure 1: 1% agarose gel electrophoresis of pCHEM4321 40 24 16 12 12 8 4 4 + |

biotechnology lab class :1. The bacterial streaking on antibiotic containing agar plates has been completed and the plates will be available for you to inspect in the lab class.2. You will complete a restriction enzyme digest on the four available plasmid stocks and then examine the products by agarose gel electrophoresis. Photographs of the gel are attached to identify the plasmid present in each analysed sample.Now consider the following questions :- For each of the four plasmids used in the practical, identify the size (in kb) of the linear DNA fragment(s) that you would expect to obtain if the EcoRI digest is complete.- Using the provided negative image of the gel, which shows the bands present in the ND and D samples for each plasmid, identify the plasmid that was present in each of the four plasmid stock.- Explain how you were able to identify plasmid in each sample using the gel, considering how linear DNA fragments of different length are separated by agarose gel…

EcoRI 1.1kbp 1.5 kbp Hindll Hindll 0.15 kbp amp 0.25 kbp EcoRI If you were to digest this plasmid with HindllI, how many fragments would be visible using gel electrophoresis? O 1 O 4

Attached is an image of EcoRI-digested plasmid samples (pBSK and pEMBL1.9); explain why the bands are found at the top, the purity of the results, and how we couple improve our results.

How does SYBR green work in DNA imaging and why does the uncut plasmid run faster than the cut plasmid? Please Explain. Thank you

In DNA purification, explain how the chromosomal DNA is separated from the plasmid DNA? Be sure to mention the specific buffer components that facilitate this process.

You have another circular plasmid. Complete and effective digestion of this plasmid with a restriction enzyme yields three bands: 4kb, 2kb, and 1 kb. In comparing the band intensity on an ethidium bromide-stained gel, you notice that the 4 kb and the 2 kb bands have the exact same brightness. The 1 kb band is exactly one fourth as bright as each of these. (Assume there is uniform staining with ethidium bromide throughout the gel.) How many times did the enzyme cut the plasmid? What is the size of the plasmid? Justify your answers to a and b above using a clearly labeled diagram showing the relative location of the cut-sites on the plasmid.

DNA Extraction by Alkaline Lysis Procedure: 1. Spin 1.5 ml of cells in a microcentrifuge at maximum speed (12,000 rpm) for 20s to pellet. Remove the supernatant completely with a Pasteur pipet or a plastic pipettor tip. The spins can be performed at 4C or at room temperature. Longer spins make it difficult to resuspend cells. 2. Resuspend pellet in 100µl GTE solution and let sit 5 min at room temperature. Be sure cells are completely resuspended. 3. Add 200µl NaOH/SDS solution, mix by tapping tube with finger, and place on ice for 5 min. 4. Add 150µl potassium acetate solution and vortex at maximum speed for 2s to mix. Place on ice for 5-15 min. Be sure mixing is complete. 5. Spin 3 min at 12,000 rpm to pellet cell debris and chromosomal DNA. 6. Transfer 0.4 ml supernatant to a fresh tube, mix it with 0.8 ml of 95% ethanol or 0.4 ml isopropanol, and let sit 2 min at room temperature precipitate nucleic acids. 7. Spin at 12,000rpm for 3 min at room temperature to pellet plasmid DNA and…

Practice question #3 chap. 2 p. 145 3. What do you think happened? How do you explain this result? Your supervisor gives you an E.coli stain (strain PIP) with an F plasmid on which is coded resistance for ampicillin. Your E.coli strain (strain ZAZ) is F- for the plasmid, and you would really like your strain to have ampicillin resistance. To favour conjugation, you grow both strains together for 2h (in broth) and plate agar containing ampicillin to grow single colonies overnight. grow overnight Inoculate agar plate containing ampicilline (amp plates) ZAZ colonies are blue, PIP colonies are yellow. Grow ZAZ and PIP together for 2h Figure 1. Schematic representation of the experimental plan. In the morning, you find multiple colonies growing on your ampicillin resistant agar plates. Because you had mixed both strains in the same culture, you needed to be able to distinguish between which colony is a ZAZ or a PIP colony growing on your plate. Fortunately, your ZAZ strain grows blue on the…

DNA and Protein Synthesis Retake Test Using the chart below, what type of mutation would take place if the highlighted and underlined "A" was deleted?: What amino acid sequence would be coded for with this mutation? What would be the affect of this mutation on the organism? i DNA AAT СТА GTA MRNA UUA GAU CAU Amino acid Leu Asp His :: point mutation :: frame shift mutation :: no affect on organism :: affect on organism : Leu - Asp - His :: Leu - Asp - Asp : Stop - lle - ? : Tyr - Met -? 18 19 20 21 22 23 24 25 96

Preforming a "blue-white screen" 3) Would bacteria that have taken up a plasmid into which a DNA fragment has been inserted, form a blue colony or a white colony when grown on this medium? Briefly explain why these bacteria would form a colony of the color you chose.

A 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.

Fluorescence confocal microscopy (FCM) - STK38 monoclonal antibody (M01), clone 2G8- 1F3. a. b. 200 μm What is the basic principle of image formation using this microscopy technique? What can be observed and concluded from the image of the specimen?

DNA Isolation A. What is “cell lysis” and why would you want to lyse cells when doing a DNA extraction? B. What do the “proteases” like meat tenderizer do to the DNA? C. What two ingredients help precipitate the DNA out of solution?

Plasmid Mapping (what is it? why is it important?).

DNA Extraction by Alkaline Lysis Procedure: 1. Spin 1.5 ml of cells in a microcentrifuge at maximum speed (12,000 rpm) for 20s to pellet. Remove the supernatant completely with a Pasteur pipet or a plastic pipettor tip. The spins can be performed at FC or at room temperature. Longer spins make it difficult to resuspend cells. 2 Resuspend pellet in 100pul GTE solution and let sit 5 min at room temperature. Be sure cells are completely resuspended. 3. Add 200ul NaOH/SDS solution, mix by tapping tube with finger, and place on ice for 5 min. 4. Add 150ul potassium acetate solution and vortex at maximum speed for 2s to mix. Place on ice for 5-15 min. Be sure mixing is complete. 5. Spin 3 min at 12,000 rpm to pellet cell debris and chromosomal DNA 6. Transfer 0.4 ml supernatant to a fresh tube, mix it with 0.8 ml of 95% ethanol or 0.4 ml isopropanol, and let sit 2 min at room temperature precipitate nucleic acids. 7. Spin at 12,000rpm for 3 min at room temperature to pellet plasmid DNA and…

Calculate these for 1 ug of plasmid DNA in a final total volume of 20 ul. All enzymes being used here are at a concentration of 10 U/µL. For double digests, give the appropriate volume for each enzyme and decide which buffer to use. (HINT: look up double digest conditions on the NEB website.) You should always calculate all volumes in advance to ensure the correct working concentrations and so that you can prepare the digests as efficiently as possible. It is a good idea to check off each component as it is added to the microfuge tube. Complete the table below, including the volume of DNA sample(s) you will need for 1 pg of the DNA (from two different methods in Experiment #3) based on the concentration(s) you determined from the OD260 value, which you will restriction digest with each of the restriction enzymes singly and in double digests in Part A. For example, the volume of DNA sample that contains 1 µg DNA for DNA sample from Part A, Experiment 3: OD260 = 0.024, dilution 500x…

Help Please Supercoiled DNA migrates through an agorose gel faster than similarly sized linear DNA? True or False? Using the plasmid map, indicate where (bp region on plasmid) these enzymes cut on the plasmid. EcoR1 1. 1 Hindlll 2. 637 Bpu101 3. 14 BamHI 4. 256 To sequence a DNA plasmid, you need 1 µg of DNA. If your DNA concentration was 0.45 µg/µL. How many µl of your DNA 0.45 µg/µL is required to get 1 µg final? Answer in µl using 2 decimal places. Your Answer: Answer units >

Part E - Exploring parallel sheet structure and hydrogen bonding patterns A different segment of a 3 sheet found in GFP has two parallel strands. The hydrogen bonding between parallel sheets, also visible in the pdb by selecting the parallel sheet view, is shown. First, locate the N- and C-termini in this image. Next, look for a pattern in the hydrogen bonding in parallel 3 sheets Select the parallel sheet view in the pdb file. Complete the sentences describing the relationship between the two strands making up the sheet. Match the words in the left column to the appropriate blanks in the sentences on the right.

Preparing plasmid DNA (double stranded, circular) for Sanger sequencing involves annealing a complementary, single-stranded oligonucleotide DNA primer to one strand of the plasmid template. This is routinely accomplished by heating the plasmid DNA and primer to 90°C and then slowly bringing the temperature down to 25°C. Why does this protocol work? What enzyme is used and what other components are required in the sequencing reaction? How does the Sanger method determine the sequence?

2. Restriction Enzyme Mapping - use any resources to assist you including the hints below. A circular plasmid molecule (12,000 total base pairs in size) was cut with a series of restriction enzymes and the digestions were size fractionated by agarose gel electrophoresis. Some digestions involved just one enzyme (single digest), some combinations of two enzymes (double digests), and one utilized all three enzymes (triple digest). Agarose gel electrophoresis of the digestions produced bands of the following sizes. Enzyme EcoRI Hindi!! Pstl EcoRI and Hindill EcoRI and Psti Hindill and Pstl EcoRI, Hindill, and Pst I Bands of the Agarose Gel (size in base pairs) 2300 and 9700 4000 and 8000 12,000 800, 1500, 2500, and 7200 2300, 3200, and 6500 4000 800, 1500, 2500, 3200, and 4000 I Draw a plasmid map showing the location of the restriction enzyme sites relative to each other for each map. Include all 7 maps in your answer.

You are provided with cultures of E. coli cells that contain the plasmid pUC118. Each student should carry out the plasmid isolation procedure. Note the appearance of the suspension after steps (v), (vi) and (vii). The steps are  (v). Resuspend the bacterial pellet in 300 μl solution A [25 mM Tris-HCl (pH 8.0) 10 mM EDTA. Ensure that the suspension is homogeneous. (vi). Add 300 μl solution B [1% (w/v) SDS 0.2 M NaOH] and mix thoroughly. (vii). Add 300 μl solution C [3M potassium acetate, (pH 4. 3)], mix thoroughly, and incubate on ice for 5 minutes.

ull rain LTE 11:53 © 1 0 A 10% Done #1 Mol Bio Restriction Analysi... Complete the following problems. Restriction enzymes (REs), which cut D NA at specific sequences, are classic tools in molecular biology. Because of their specificity in cutting DNA, REs can be used to "map" DNA sequences by analyzing the fragments generated upon restriction digest, as in the example shown in Figure 1. Your task is to study the circular plasmid, pMBBS, through restriction digests. You subjected the PMBBS plasmid to complete digestion by different combinations of three REs (EcoRI, BamHI, and Xhol), and analyzed the results on an agarose gel, shown below. Using the data you can glean from this gel, answer the questions that follow. EcoRI BamHI Xhol DNA size ladder 600 bp 500 bp 400 bp 300 bp 200 bp 100 bp VC 100bp Plus DNA ladder from Vivantis Technologies *The same total amount of DNA was loaded in each lane. 1. What is the total size of the PMBBS plasmid in bp? Answer: bp 2. How many cut sites on the…

You will be setting up your diagnostic digest on three plasmids, the ones you began with, pGFPuv and PHSG298 and your presumptive pHSG298-GFP. Complete the table below: Solution Stock Working Volume for one reaction Volume for Master Mix (3.5X) Cutsmart buffer 10X 1X Restriction enzyme(s) 1 µl uL Water UL UL 2 µl 25 pL DNA Total volume

In bacterial transformation ... 1, Why do you have to incubate the competent bacterial cells on ice after the addition of plasmid DNA? 2. Why do you have to add SOC (Super Optimal Broth with Catabolite repression) medium to bacterial cells? 3. Why do you have to incubate the bacterial cells in SOC medium at 37oC for 45 minutes?

4. Look at the gel image and answer the questions below and be specific. a) Based on your calculations of the DNA concentrations, how much DNA was loaded into each well? Do you see DNA for each of your samples? If not, why do you think that is so? b) Is the DNA in a single sharp band, multiple bands or a smear? What would each of these scenarios be due to, and why would you see them for your samples? c) Do you see multiple bands in your plasmid DNA sample? What are they?

Gel Electrophoresis A. Why does DNA travel toward the positive electrode in the gel chamber? B. What is a ladder sequence and why is it useful to include in a gel?