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25 Nov

Practical Exercise | Good Grade Guarantee!

1BCH30003/BCH80001Practical ExerciseIsolation and analysis of plasmid pAB2 from E. coliINTRODUCTIONIn this exercise, a recombinant plasmid pAB2 constructed from parent (non-recombinant)plasmid vector pTZ18U was isolated from Escherichia coli (E. coli) cells. The parent plasmidpTZ18U (NCBI Accession No. L37352.1) is 2860 bp in size and has multiple cloning sitesfor cleavage by different restriction endonucleases, including a single site for the restrictionendonuclease EcoRI (see Figure 1). pAB2 (kindly donated by Dr Tony Barton) wasconstructed by cloning a fragment of baculovirus DNA into an EcoRI-digested pTZ18U.Figure 1. Map of parent plasmid vector pTZ18U showing the EcoRI restriction site (adaptedfrom www.addgene.org)An EcoRI digestion of baculovirus (Autographa californica nucleopolyhedrovirus) DNA(NCBI Accession No. NC_001623.1) produced 52 fragments. The recombinant plasmidpAB2 was constructed by cloning one of these 52 fragments into an EcoRI-digested pTZ18Uparent plasmid.2In this practical, you will determine which of the 52 DNA fragments was inserted intopTZ18U to obtain pAB2. The practical will be performed in two parts. In Exercise 1, youwill isolate recombinant plasmid DNA from E. coli cells and digest the isolated plasmid DNAwith a restriction enzyme followed by gel electrophoresis to estimate the size of the insert. InExercise 2, you will perform in silico restriction digestion of pAB2 and baculovirus toestimate which one of the 52 fragments was inserted into the parent plasmid.EXERCISE 1: ISOLATION AND RESTRICTION DIGESTION OFRECOMBINANT PLASMID DNAMATERIALS Frozen pellet of E. coli cells transformed with recombinant plasmid AB2 (pAB2) whichcontains an insert of the baculovirus DNA Wizard® Plus SV Minipreps DNA Purification kit (Promega) containing; cell resuspensionsolution, cell lysis solution, alkaline protease solution, neutralization solution, washsolution, nuclease-free water, spin columns and collection tubes. The DNA purificationkit will be ready for you in a tray. Restriction enzyme HindIII: 10 units/µL (10U/ µL) Restriction enzyme buffer, i.e., HindIII bufferMETHODSA. Preparatory StepThis will have been performed for you by the lab staff: An overnight culture of transformedE. coli cells containing the recombinant plasmid pAB2 was obtained, and 3mL was spundown so that the cells form a pellet. The frozen pellet will be supplied to you, and this shouldbe used as your starting sample.B. Isolation of DNA of the recombinant plasmid pAB2You will be using the Wizard® Plus SV Minipreps DNA Purification kit (Promega) forisolation of plasmid DNA. You should follow the instructions (abbreviated protocol) givenhere. All material used in this step is from the kit. You should also read (for your interest) thetechnical bulletin on Blackboard which describes this system.B.1 Production of cleared cell lysate1. Resuspend the frozen E. coli cell pellet provided to you into 250 µL of cell resuspensionsolution and mix well.2. Add 250 µL of cell lysis solution and mix well by inverting the tube a few times.3. Add 10 µL of alkaline protease solution and mix well by inverting the tube a few times4. Incubate the tube at room temperature for 5 min5. Add 350 µL of neutralization solution and mix well by inverting the tube a few times6. Centrifuge the tube at 14,500 rpm for 10 min3B.2 Binding of plasmid DNA1. Insert the spin column (with the supernatant) into a collection tube2. Transfer the supernatant (cleared lysate) into a spin column (avoiding the pellet)3. Centrifuge at 14,500 rpm for 1 min and discard the flowthrough (liquid in the collectiontube, NOT the spin column)4. Re-insert the spin column in the same collection tube.B.3 Washing1. Add 750 µL of wash solution (ethanol added) to the spin column2. Centrifuge at 14,500 rpm for 1 min and discard the flow-through, i.e., the liquid in thecollection tube, NOT the spin column! The column has your plasmid DNA bound to it!3. Re-insert the spin column in the same collection tube4. Add 250 µL of wash solution (ethanol added) to the spin column5. Centrifuge at 14,000 rpm for 2 min and discard the collection tube (including theflowthrough, NOT the spin column)6. Transfer the spin column to a sterile 1.5 mL microfuge tube (please take care that the spincolumn does not contain any wash solution)B.4 Elution1. Add 50 μL of Nuclease-Free Water to the spin column2. Centrifuge at 14,000 rpm for 1 min3. Discard the spin column and store the 50 μL plasmid DNA (eluted in the microfuge tube)on ice for the next step: restriction digestion: see below.C. Digestion of plasmid DNA with restriction enzyme1. This section requires your full attention, so read carefully first and ask any questions.2. Hold the restriction enzyme tube on ice all the time. DO NOT leave it without ice for anylength of time, as these enzymes are highly thermo-sensitive and VERY EXPENSIVE,and other students need to use the same tube.3. Set up two microfuge tubes ON ICE, and add the reagents in the order given in the table.4. ABSOLUTELY ENSURE changing the tip each time while doing the additions, or youwill contaminate everything.
Tube No.
Plasmid DNAfrom the aboveeluted prep
Restriction enzymeHindIII (10U/1 µL)
2 µL
15 µL
2 µL
1 µL (10U)
2 µL
16 µL
2 µL
0 µL
5. Incubate the above tubes in a waterbath set at 37ºC for 45 min.6. Start preparing the gel for electrophoresis (next section) during the incubation time.7. After incubation, add 5 µL of the provided gel loading dye to both the tubes, mix welland store on ice until loading these samples onto the gels (see below).4D. Gel electrophoresis1. Prepare 300 mL of 1x TAE electrophoresis buffer by diluting the given 10x TAE buffer.For this, you will have to add 270 mL of distilled water to 30 mL of 10x TAE buffer.2. Seal the ends of the gel-casting tray masking tape and place a 8-well slot former (comb)onto the gel-casting tray about 1 cm from the end.3. Weigh out 0.3 g of agarose powder directly in a 100 mL conical flask and add 30 mL of1x TAE buffer to it and heat it in a microwave for 60 seconds.4. Using heat-resistant gloves, CAREFULLY take the flask out of the microwave, hold itagainst light swirl it a little to see if all the agarose has melted to form a clear solution, orare any particles seen. Otherwise heat the flask for another 10-20 seconds until all particlesdissolve. Then cool the gel solution to 50-55ºC (cool enough to be able to place your handon the side of the flask for a few seconds).5. When the gel solution has cooled, add 3 µL of the Gel Red staining agent to it.6. Pour the gel solution gently, along a side, into the tray. Do not allow any air bubbles toform as you pour the gel solution into the tray; if these do form, prick them quickly with aclean pipette tip. Let the gel set (solidify) for about 15-20 minutes.7. Once the gel has set, remove the masking tape and gently remove the comb. Transfer thegel tray into the gel tank. Pour the remaining 1x TAE buffer (approximately 250-270 mL)into the gel tank. The volume should be just enough to cover the gel, but not spill.8. Load four separate wells in the gel as follows: (i) tube 1 above: your 10 µL restrictiondigest (ii) tube 2 above: your 10 µL digest no-enzyme control; (iii) 4 µL of DNA molecularweight marker (GeneRuler), and (iv) 5 µL of EcoRI-digested pAB2 (100ng/µL) (providedto you).9. As soon as you finish loading, ask the demonstrator to turn on the electrophoresisequipment. The gel should be electrophoresed at 120 V for about 30-45 min.10. At the end of the electrophoresis, ask the demonstrator to turn off the equipment.Then carefully remove the gel tray and slide the gel into a press‐seal plastic bag.11. Inspect the gel on the UV‐light box. BEWARE OF UV RADIATION! Capture animage of the gel using the gel documentation camera, AND SAVE IT WITH YOUR ANDYOUR PARTNER’S NAME AND PRAC DATE AND TIME.12. Dispose of the electrode buffer carefully down the sink and wash the apparatus.13. Save the image file on a memory stick or email it to yourself from the hard drive. YOUNEED THIS FOR YOUR REPORT, SO DON’T LEAVE WITHOUT IT: WECANNOT HELP YOU AFTERWARDS.14. YOU MUST SHOW YOUR SAVED GEL IMAGE TO THE DEMONSTRATORAND THEY MUST MAKE A RECORD THAT YOU HAVE SHOWN IT. Thisimage may be used while marking your report, so it is important that you ensure thishas been done.5EXERCISE 2: IN SILICO ANALYSIS OF RECOMBINANT PLASMID DNAA. Restriction Digestion of baculovirus DNA insert in a plasmid1. Click on this link to use the NEBCutter tool: http://nc2.neb.com/NEBcutter2/2. Enter the GenBank number NC_001623.1 and select plasmid vectors and viral + phageas standard sequences. Please remember that the sequence is circular. Click submit.3. Click Custom Digest under Main options4. Tick EcoRI and click Digest5. Click Fragments under List6. Print the list of fragments and attach this as an appendix.7. Find out the fragments with appropriate sizes that may have been possibly be cloned intothe pTZ18U vector to get pAB2 (Hint: find the fragments with sizes that match your bandsize from the gel image. Note that gels do not give you exact size, and therefore, you mayhave 2-3 candidates and will have to identify the most appropriate one later)8. Note down the co-ordinates of all the appropriate fragments in the baculovirus sequence,as these will be used later to find the sequence of the insertB. Determination of baculovirus fragment sequence – FASTA format1. Click on this link: https://www.ncbi.nlm.nih.gov/2. Select All Databases and search for NC_001623.13. Click on FASTA and copy the FASTA sequence in a notepad file and save asNC_001623.1_FASTA4. On the top right corner, you will find Change region shown, enable ‘Selected region’ andenter the co-ordinates for one of the fragments from Step A.8 (Exercise 2) and click UpdateView.5. Copy the FASTA sequence in a word file, select all text, change the font to ‘Courier New’and save as NC_001623.1_Fragment 16. On the top right corner, you will also find Customize view, under Display options enableShow reverse complement and click Update View7. Copy the FASTA sequence in a word file, select all text, change the font to ‘Courier New’and save as NC_001623.1_Fragment 1Rev8. Repeat steps 1 to 7 for all the appropriate fragments and their reverse complements. Pleaseremember to disable Show reverse complement before you start this process for thesecond fragment.C. Determination of parent plasmid pTZ18U sequence – FASTA format1. Click on this link: https://www.ncbi.nlm.nih.gov/2. Select All Databases and search for L37352.13. Click on FASTA and copy the FASTA sequence in a word file, select all text, change thefont to ‘Courier New’ and save as L37352.1_FASTA6D. Construction of recombinant plasmid to identify the correct insert1. Open the notepad file L37352.1_FASTA2. Find the EcoRI site. This can be done by finding the sequence GAATTC3. Press enter after G from the sequence and copy and paste the composite sequenceNC_001623.1_Fragment 14. Use ‘Save As’ to save this new word file as pAB15. Repeat steps 1 to 4 for all composite sequences i.e. NC_001623.1_Fragment 1Rev,NC_001623.1_Fragment 2, NC_001623.1_Fragment 2Rev and so on.6. Save As these word files as pAB1Rev, pAB2, pAB2Rev and so on.E. Identification of the correct insert1. Copy the sequence from the word file pAB12. Click on this link to use the NEBCutter tool: http://nc2.neb.com/NEBcutter2/ and paste thesequence in the DNA sequence window3. Please remember that the sequence is circular. Click submit.4. Click Custom Digest under Main options5. Tick HindIII and click Digest6. Click Fragments under List7. Print the list of fragments and include this in your reports as a table for the compositesequence for first possible insert from baculovirus. Present this analysis and map of theproposed structure of pAB2 and your evidence for the proposal7. Repeat the steps 1 to 7 for all the other word files i.e. pAB1Rev, pAB2, pAB2Rev and soon. Please remember to print the list of fragments for each of the possible inserts8. Compare the fragments that you get for each insert and their reverse complements with thegel image you have obtained after plasmid digestion and estimate the correct insert.LAB ASSESSMENTo Each lab assessment in this unit will be worth 40 raw marks.o Lab Report: 40 raw marks. Must be no more than 3000 words, including references. Follow the specific requirements for this report, given below in the table. Please arrange as first page (title, names etc.), then the report, then the feedback sheet(next page). Submit as one single document in Turnitin (DO NOT email, or upload part by part!)7LAB REPORT REQUIREMENTS
6 marks
1. What are plasmids? Why are plasmids used as cloning vectors?
2. Give information about the pTZ18U plasmid (provide figure ifapplicable)
3. Give information about the baculovirus
4. How was pAB2 generated? (provide figure if applicable)
Aims: (1 mark)5. Aims of the practical exercise
1 mark
Materials and Methods
4 marks
6. Plasmid extraction
7. Plasmid digestion
8. Gel Electrophoresis
9. In silico analysis
14 marks
10. Plasmid extraction and digestion (provide gel image)
11. Fragments of baculovirus, potential structures of pAB2
12. Comparison of gel image with potential plasmid digests to identify thecorrect insert
10 marks
13. Discuss your gel and bands that you obtain on the gel
14. Identify the correct insert and explain the logic behind your conclusion
15. Discuss any discrepancies in the gel and suggest reasons
2 marks
3 marks
UPLOAD on Blackboard. Look under Assessment and find the turnitin symbolfor ‘Practical 1 reports’.Due date: one week from your own practical date, 23:59 pm of the due date. e.g.,for pracs any time on March 8: due date 23:59 pm, March 15.Refer to the Laboratory Manual for all detailed instructions.

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