### Understanding Plasmid Mapping and Fragment Analysis#### 1. Gel Electrophoresis of pBR322 Digested with SalI, BamHI, and PstIThis diagram depicts the circular DNA plasmid map of pBR322, which is 4361 base pairs (bp) in length. Various restriction enzyme sites are denoted, showing where specific enzymes can cut the plasmid DNA.- **Restriction Enzymes and Their Sites**: - **PstI**: Cuts at 3607 bp. - **BamHI**: Cuts at 375 bp. - **SalI**: Cuts at 651 bp. - Other sites include HindIII, EcoRI, EcoRV, and NdeI, specified with their respective cut positions (e.g., HindIII at 29 bp).When pBR322 is cut with the enzymes SalI, BamHI, and PstI, it generates specific DNA fragments. Students are expected to map these cuts and calculate expected fragment sizes.#### 2. Expected Size of Cloned Plasmid with Insert- **Cloning Strategy**: - To clone an insert of 500 bp using BamHI and SalI, first treat pBR322 with both enzymes. - Calculate the expected final size of the plasmid with the insert: - Remove the BamHI-SalI fragment. - Insert the 500 bp piece.This educational exercise involves understanding restriction mapping, digestion, and calculating fragment sizes for successful molecular cloning.

DNA or proteins are separated via gel electrophoresis based on their molecular weight. It makes use…

1. Draw a gel showing the bands/fragments generated from cutting the plasmid below with Sall, BamHI and Pstl. Make sure you label the bands with the expected sizes. PstI. 3607 3000 4000 amp ori HindIII EcoRI EcoRV 4359 0 29 185 pBR322 4361 bp 2295 | NdeI 375 tet 2000 BamHI 651 Sall 1000 2. If you want to use the plasmid above to clone an insert of 500 bp using BamHI and Sall, what would your expected FINAL plasmid+insert size to be if your cloning was successful? (remember that you first have to treat the intact pBR322 with BamHI and Sall to prepare it for introducing the insert).

1. Draw a gel showing the bands/fragments generated from cutting the plasmid below with Sall, BamHI and Pstl. Make sure you label the bands with the expected sizes. PstI. 3607 3000 4000 amp ori HindIII EcoRI EcoRV 4359 0 29 185 pBR322 4361 bp 2295 | NdeI 375 tet 2000 BamHI 651 Sall 1000 2. If you want to use the plasmid above to clone an insert of 500 bp using BamHI and Sall, what would your expected FINAL plasmid+insert size to be if your cloning was successful? (remember that you first have to treat the intact pBR322 with BamHI and Sall to prepare it for introducing the insert).

Human Anatomy & Physiology (11th Edition)

11th Edition

ISBN:9780134580999

Author:Elaine N. Marieb, Katja N. Hoehn

Publisher:Elaine N. Marieb, Katja N. Hoehn

Chapter1: The Human Body: An Orientation

Section: Chapter Questions

Problem 1RQ: The correct sequence of levels forming the structural hierarchy is A. (a) organ, organ system,...

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Hind III' Hind III .9 kb .9 kb Bam HI plasmid A .9 kb .7kb Bam HI .3 kb plasmid B .9 kb .4 kb .3 kb .5 kb/ Eco RI Bgl Il .4 kb Eco RI .7 kb .5 kb -Bgl II Bam HI Eco RI You requested that a lab run by a friend of yours share a couple of plasmids that they created with you. She was willing to send you the ones that you asked for in exchange for citation in any papers that were published using the plasmids. Eco RI However, the labels from two vials have fallen off in the shipping because the adhesive used to hold them was not able to withstand the temperature, being shipped on dry ice. Each vial contained a different plasmid and now you do not know which plasmid is in which. Fortunately, you have restriction maps for both plasmids, shown above. Explain how you would determine which vial contained each plasmid and give a graphical representation of your expectations related to your method of Bam HI discovery. (25pts)
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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2. pBR3322, an artificially made plasmid, is shown below. ECORI Hindill BamHI Pstl Amp ORI ↓↓ Tetr pBR322 (4.36 kb) ↑ Pvull Xmalll Describe 3 features that this plasmid has and indicate each one on the diagram. i) ii) 111]