Introduction to Vectors

1
Introduction to Vectors

• In order to study a DNA fragment (e.g., a gene),
  it needs to be amplified and eventually purified.
• These tasks are accomplished by cloning the DNA
  into a vector.
• A vector is generally a small, circular DNA
  molecule that replicates inside a bacterium such
  as Escherichia coli (can be a virus).

Ch. 1-1
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Cloning Scheme
Digest
Ligate
Amplify and Prep
1-1
3
Vector Types

• There are three commonly used types of vectors
• 1) plasmid vectors (e.g., pUC plasmids)
• 2) bacteriophage vectors (e.g., phage ?) and
• 3) phagemid vectors (e.g., pBlueScriptTM).
• Each has a different use, and there are many
  derivatives of these basic building blocks. In
  BRITE, you will be using plasmids (phagemids).

Ch. 1-1
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Plasmids

• Circular DNA molecules found in bacteria
• Replicated by the hosts machinery independently
  of the genome. This is accomplished by a sequence
  on the plasmid called ori, for origin of
  replication.
• Some plasmids are present in E. coli at 200-500
  copies/cell

Ch. 1-1
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Plasmid Engineering

• Plasmids also contain selectable markers.
• Genes encoding proteins which provide a selection
  for rapidly and easily finding bacteria
  containing the plasmid.
• Provide resistance to an antibiotic (ampicillin,
  kanamycin, tetracycline, chloramphenicol, etc.).
• Thus, bacteria will grow on medium containing
  these antibiotics only if the bacteria contain a
  plasmid with the appropriate selectable marker.

Ch. 1-2
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Safety Features

• Modern cloning plasmids have been engineered so
  that they are incapable of transfer between
  bacterial cells
• Provide a level of biological containment.
• Naturally occurring plasmids with their
  associated drug resistance genes are responsible
  for the recent rise in antibiotic-resistant
  bacteria plaguing modern medicine.

Ch. 1-2
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Transforming plasmids Into bacteria
Ch. 1-2
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Screening for Inserts
1-3
9
Size of the cDNA insert?
cDNA Insert
Ch. 1-4
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Vector Preparation

• In order to use a vector for cloning, sequencing,
  etc., it is necessary to isolate the vector in a
  highly purified form.
• Routinely done by most labs.
• Many companies now sell kits which provide all
  the solutions necessary for preparing DNA.
• Based on similar procedures

Ch. 1-4
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Grow the bacteria

• Grow an overnight (ON) culture of the desired
  bacteria in 2 ml of LB medium containing the
  appropriate antibiotic for plasmid selection.
  Incubate the cultures at 37C with vigorous
  shaking.

Ch. 1-6
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Naming your clones
School 0- Rutgers Univ. 1- Bayonne 2-
Bordentown 3- Bridgewater-Raritan 4- Colonia 5-
East Brunswick 6- Franklin 7- Hillsborough 8-
James Caldwell 9- JFK Memorial 10- JP Stevens 11-
Monmouth 12- Montville 13- New Brunswick 14-
Pascack Hills 15- Pascack Valley 16- Pingry 17-
Rutgers Prep. 18- Watchung Hills 19- West
Windsor-Plains.
Year
School
0AV06-12
Your initials
Clone
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1. Plasmid MiniPreps

• Obtain your overnight cultures Bacteria grown
  in 2 ml of LB medium containing the appropriate
  antibiotic for plasmid selection.
• This culture was incubated overnight at 37C
  with vigorous shaking.

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2. Transfer the cells to a tube and centrifuge

• Transfer 1.5 ml of the culture to a microfuge
  tube and pellet the cells for 1 minute at full
  speed (12,000 rpm) in the microcentrifuge.
• First tap or gently vortex the glass culture
  tube to resuspend the cells which have settled.
  The culture can be transferred to the microfuge
  tube by pouring.

Ch. 1-6
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2b. Remove the supernatant

• Remove the growth medium (supernatant or sup) by
  aspiration or by using the P-1000.
• Leave the bacterial pellet as dry as possible so
  that additional solutions are not diluted.

Ch. 1-6
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3. Resuspend the cell pellet

• Resuspend the bacterial pellet in 150 µl of
  Buffer Solution I by vigorous vortexing.
• Add 150 ml of Solution I, cap the tube, and
  vortex on the highest setting (pipetman can be
  used). Look very closely for any undispersed
  pellet before proceeding to the next step. It is
  essential that the pellet be completely
  dispersed.
• Solution I contains three essential components
  Glucose and Tris are used to buffer the pH of
  the cell suspension.
• EDTA is a chemical that chelates divalent
  cations (ions with charges of 2) in the
  suspension, such as Mg. This helps break down
  the cell membrane and inactivate intracellular
  enzymes.

Ch. 1-6
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4. Add Solution II

• Add 150 µl of Solution II, mix gently 10-15
  times.
• Close the tube tightly and mix the contents by
  slowly inverting the tubes five times. During
  this step a viscous bacterial lysate forms (the
  cells lyse). Do not vortex!! This will shear the
  DNA and contaminate your DNA preps.

Ch. 1-7
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5. Add Solution III

• Add 300 µl of Solution III. Mix gently 10-20
  times.
• Mix by inverting the tubes several times. Do not
  vortex. A white precipitate consisting of cell
  debris and SDS will form.

Ch. 1-7
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6. Centrifuge cell debris

• Centrifuge for 5 minutes at full speed in the
  microcentrifuge.
• A white pellet will form on the bottom and side
  of the tube after centrifugation. During this
  centrifugation step, place the necessary number
  of spin columns into the respective number of 2
  ml Collection Tubes and label each appropriately.
  This is also an ideal time to label 1.7 ml
  microcentrifuge tubes for use in the final step
  to collect the miniprep DNA.

Ch. 1-7
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7. Transfer the sup (DNA) to spin column.

• Using a P-1000 set at 600ul, transfer the
  supernatant to the appropriately labeled spin
  column which has been inserted into the 2 ml
  microcentrifuge tube.
• Do not contaminate the spin column with the
  white precipitate.

Ch. 1-8
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8. Centrifuge the spin column

• Centrifuge for 1 minute at full speed, and drain
  the flow-through from the collection tube.
• In a single action, remove the spin column from
  the 2 ml Collection Tube and pour the
  flow-through, or liquid that passed through the
  column, into the waste container. Place the spin
  column back into the 2 ml Collection Tube.

Ch. 1-8
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9. Wash the column

• Add 400 ul of Wash buffer to the spin column
  contained in the 2 ml Collection Tube, centrifuge
  at full speed for 1 minute, and drain the
  flowthrough.
• This buffer helps to further remove any nucleases
  that may have co-purified with the DNA. Remove
  the liquid that has passed through the column in
  the same way as performed in Step 9.

Ch. 1-9
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10. Spin the column

• Place the AB spin column in a fresh 1.7 ml
  microcentrifuge tube (with lid cut off) and
  centrifuge again for 1 minute at full speed to
  remove any residual wash solution that might
  still be in the column.
• Any residual wash solution must be removed
  because the ethanol contained in this solution
  may interfere with further DNA manipulations. It
  is normal to remove a small amount of liquid from
  the column at this step, however if a significant
  amount of solution (50-100 ul or greater) is
  found in the collection tube, repeat this step.

Ch. 1-9
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11. Elute the DNA

• Place the spin column into an appropriately
  labeled 1.7 ml microcentrifuge tube and add 50 ul
  of sterile waterto the column. Centrifuge at full
  speed for 1 minute.
• This is the final step and elutes or removes the
  plasmid DNA from the column and back into
  solution so that it collects in the
  microcentrifuge tube. This 50 ul of solution
  contains your plasmid DNA

Ch. 1-9
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Store your DNA

• Remove the spin column from the labeled 1.7 ml
  microcentrifuge tube and close the lid on the
  tube tightly.
• Store the miniprep DNA in your freezer box
  (-20C).

26

• Plasmid Problem Set
• 1. What are vectors used for?
• 2. What is a polylinker?
• 3. What is a selectable marker?
• In preparing double-stranded plasmid DNA preps
• The first step of the prep is to centrifuge the
  culture. Do you want to save the supernatant or
  the pellet?
• Name two functions for Solution II.
• Why does the cell suspension become viscous after
  adding Solution II ?
• After you add the cell lysis supernatant to the
  spin column and spin do you want to save the
  liquid in the bottom of the collection tube or
  the column?
• After you add the 50 ul of water to the spin
  column and centrifuge do you want to save the
  column or the liquid in the bottom of the tube?