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Introduction to Laboratory Science > Plasmid DNA purification and Analysis > Flashcards

Plasmid DNA purification and Analysis Flashcards

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1
Q

What are the risks associated with this practical?

A
  • Live strains of E. Coli used. Tubes and pipette tips that have been in contact with the live bacteria should be placed immediately after use into the disinfectant containers provided. No liquid should be put in the sink
  • Solution 2 contains sodium hydroxide and solution 3 contains acetic acid. Wear Gloves when handling these
  • The agarose gel contains a fluorescent dye (SafeView) that binds to DNA. It is a skin irritant and so you must wear gloves when handling the gel
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2
Q

What does recombinant technology allow?

A

the molecular cloning of genes

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3
Q

What do the practical applications of recombinant DNA technology include?

A
  • identification of disease-causing genes in humans

- production of human proteins in microorganisms and tissue culture cells for use as pharmaceuticals

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4
Q

Why do we use E. coli in the lab?

A
  • it is very easy to grow and manipulate in the lab

- the K12 strains used in the laboratory are harmless

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5
Q

What are plasmids?

A

extra-chromosomal pieces of DNA found naturally in many bacteria

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6
Q

Give features of plasmids

A
  • usually circular
  • composed of double-stranded DNA
  • can replicate inside bacterial cells alongside the chromosome
  • often contains genes for one or more traits that are beneficial to the host
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7
Q

How do plasmids move between cells?

A
  • horizontal transmission - moving from cell to cell

- vertical transmission - moving to daughter cells following cell division

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8
Q

When do plasmids replicate?

A
  • once per division: low copy number

- autonomously throughout the cell cycle of the bacteria - high copy number

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9
Q

How do plasmids range in size?

A

from about 1000 DNA base pairs (bp) to up to several thousand kilobase-pairs

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10
Q

What laboratory processes can artificial plasmids be used for?

A

DNA cloning or protein expression studies

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11
Q

What are the plasmids in this practical?

A
  • pBlusecript
  • pGLO
  • both are cloning vectors
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12
Q

What are the key steps in the isolation of Plasmid DNA from e.coli?

A
  1. Harvesting a sufficient quantity of bacterial cells that harbour the plasmid
  2. Breaking open the bacterial cells
  3. Precipitating cellular debris (proteins, chromosomal DNA etc.) leaving the plasmid DNA in solution
  4. Precipitating the plasmid DNA out of solution
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13
Q

How can you get rid of RNA in your plasmid isolate?

A

adding RNAse (ribonuclease)

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14
Q

What is electrophoresis?

A

a technique used to separate molecules according to differences in molecular size, and is based on the movement of charged molecules in an electric field

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15
Q

What are agarose gels derived from?

A

agar-agar

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16
Q

What are agarose gels used for?

A

the separation of nucleic acid molecules

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17
Q

How do agarose gels separate nucleic acid molecules?

A

the matrix of the gel acts as a molecular sieve, or a matrix of holes, through which smaller DNA fragments and RNA can move more easily than the larger ones

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18
Q

Give features of the pore size of agarose gel

A
  • depends on the concentration of agarose (usually 0.7-2%) which can be varies depending on the size of the nucleic acid fragments to be separated
  • Higher percentage gels (> 1.5%) allow separation of smaller fragments (< 1 kbp), whereas lower percentage gels (<1 %) are more suitable for larger fragments (> 3 kbp)
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19
Q

Where is the agarose gel slab placed?

A

between two electrodes in a chamber and immersed in a conductive buffer solution

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20
Q

Where are individual nucleic acid samples applied?

A

to wells in the gel and an electric current is then applied

21
Q

At what pH is DNA negatively charged?

A

a pH of around 8.3

22
Q

How do fragments of the same size migrate in electrophoresis?

A

as single bands

23
Q

What does the dye SafeView do?

A

binds to DNA and RNA and that fluoresces under long-wave UV light. The gel can then be viewed under UV light and photographed

24
Q

What can agarose gel electrophoresis be used to do?

A

to estimate the size of DNA fragments and for quantifying amounts of DNA in a sample

25
Q

Why can interpreting gels containing plasmid DNA be difficult?

A
  • In addition to the plasmid DNA, contaminating chromosomal DNA and RNA may be seen
  • Plasmid DNA migration through an agarose gel depends not only on the size (molecular weight) of the plasmid, but also its molecular conformation
26
Q

What are the three conformations plasmid DNA can exist in?

A
  • supercoiled plasmid DNA
  • relaxed or ‘nicked’ plasmid DNA
  • linear plasmid DNA
27
Q

What is supercoiled plasmid DNA?

A
  • The circular double-stranded plasmid DNA usually exists in the bacterial cell as a ‘supercoiled’ molecule (the DNA molecule coils about itself to form a compact structure)
  • This form is the fastest-moving in a gel
28
Q

What is relaxed/ nicked plasmid DNA?

A
  • If one of the strands of DNA is ‘nicked’ or cut during the plasmid preparation the torsional stress of the supercoiled molecule is released so that the plasmid develops into a relaxed open circle form
  • This is the slowest-moving form of plasmid DNA in a gel, as the larger size restricts movement through the gel pores
29
Q

What is linear plasmid DNA?

A
  • Forms when both strands of the molecule are cut in the sample place – either deliberately by a restriction endonuclease or by accident during the extraction process
  • Usually migrates at a rate in-between that of the supercoiled and the relaxed forms
30
Q

What is the source material for our plasmid preparations?

A

Two overnight broth cultures A and B of E.coli that contain either pBluescript plasmid of pGLO plasmid which are different sizes.

31
Q

what can we use agarose gel electrophoresis to identify in this plasmid?

A
  1. The different molecular confirmations of plasmid isolated
  2. The different species of nucleic acid that can be observed on an agarose gel
  3. Which culture has which plasmid
32
Q

What are the experimental steps of plasmid miniprep? (short version)

A
  1. Harvest bacteria by centrifugation
  2. Resuspend bacterial pellet in solution containing glucose, Tris buffer and EDTA
  3. Lyse bacteria by adding alkaline solution containing NaOH and SDS
  4. Precipitate chromosomal DNA, proteins and lipids by adding acidic solution containing potassium acetate
  5. Pellet celular debris by centrifugation and remove supernatant containing plasmid DNA to a new microcentrifuge tube
  6. Precipitate DNA with iso-propanol and centrifuge to pellet the plasmid DNA on the bottom of the microcentrifuge tube
  7. Wash plasmid DNA pellet with cold 70% ethanol and remove ethanol after centrifugation
  8. resuspend plasmid DNA in Tris-EDTA buffer
33
Q

What is the alkaline extraction procedure of plasmids?

A
  1. Overnight broth cultures are added to a microcentrifuge tube and centrifuged at high speed for a minute to pellet the bacterial cells
  2. The broth supernatant is poured into a discard pot or disinfectant. To remove the last drops of liquid the tube is inverted in tissue paper and the contaminate tissue is discarded. The pellet must not be dislodged
  3. 200 ul ice-cold solution 1 (containing glucose, Tris and EDTA) is added to the pellet using a pipette. The pellet is dislodged from the bottom of the tube using the end of the pipette tip. The cells are resuspended in the solution by pipetting up and down.
  4. 400 ul of solution 2 (containing sodium hydroxide and SDS) is added to the mix and inverted 5 times to mix. The suspension inside the tube clears as the cells lyse. Tubes are left for a minute on the bench to allow this to happen
  5. 300 ul ice-cold solution 3 (containing a mixture of acetic acid and potassium acetate) is added and the tube is inverted 4 times. The tubes are left on ice for 5 minutes and then a white precipitate containing SDS, cellular debris from bacteria (protein and lipid) and chromosomal DNA forms
  6. tubes are centrifuges at 5 minutes at high speed to pellet the cellular debris
  7. the supernatant (containing the plasmid) is transferred to a fresh microcentrifuge tube using a pipette
  8. the centrifuge tube is now filled with approximately 500 ul iso-propanol (IPA - precipitates nucleic acids from solution). The tube is inverted to mix contents and incubated at room temperature for 2 mins
  9. tube is centrifuged for 5 minutes at high speed. The nucleic acid should now be visible as a small pellet at the bottom of the tube
  10. 1ml Ice-cold 70% ethanol is added to the DNA pellet and washed by inverting the tube once without disturbing the pellet. Centrifuged for 1 min to keep pellet in the tube
  11. All supernatant removed using a tip. All ethanol should be removed (flick the tube to ensure there is none remaining) otherwise DNA will not dissolve later. The pellet is allowed to dry at room temperature
  12. When the pellet is dry resuspend in 30 ul of TE buffer, and flick the tube to help dissolve the DNA pellet
  13. we have no extracted plasmid DNA
34
Q

When placing tubes in the centrifuge how should you place them

A
  • so that they are balanced
  • have a lid on the rotor
  • place the tubes with the hinge of the cap of the tube sticking away from the centre - means that any pellet will always form directly below the hinge in the tube
35
Q

Ice cold solution 1 contains glucose, Tris and EDTA. What do these do?

A
  • glucose increases the osmotic pressure outside the cells so that they become vulnerable to rupture
  • tris is a buffering agent added to maintain a constant pH
  • EDTA binds to ions that are needed by enzymes that can degrade DNA. This means these enzymes (DNases) are inhibited from degrading the DNA
36
Q

Solution 2 contains sodium hydroxide and the detergent SDS. What do these do?

A
  • the alkaline mixture ruptures the cells
  • NaOH also denatures the DNA (plasmid and chromosomal) into single strands
  • the detergent breaks up the lipid proteins of the cell membrane and solubilises cellular proteins
37
Q

Solution 3 contains acetic acid and potassium acetate. What do these do?

A
  • the acid neutralises the pH allowing the DNA strands to renature
  • the salt precipitates SDS from the solution along with all the the cellular debris from the bacteria to form a precipitate composed of SDS, protein and lipid. The E. coli chromosomal DNA, which is only partially renatured due to it’s large nature is also trapped in this precipitate.
38
Q

What does iso-propanol do?

A

precipitates any nucleic acids (RNA and DNA) from the solution and will also precipitate proteins after time

39
Q

What does the addition of ethanol do?

A

helps remove any remaining salts and SDS

40
Q

What does a TE buffer contain?

A

Tris and EDTA

41
Q

Why may you have trouble dissolving the plasmid pellet given that pure DNA is soluble in water?

A

Because it is mixed with other cellular components

42
Q

Why percentage agarose gel would be suitable to use for our experiment to see the plasmid DNA?

A

0.8% agarose gel

43
Q

how far above the gel should the level of electrophoresis buffer be?

A

About 2mm

44
Q

What are molecular weight markers used for?

A

to calibrate the gel and serve as standards to estimate the size of the unknown DNA fragments

45
Q

What is the positive control we use for this experiment? (when running the electrophoresis)?

A

Previously purified pBluescript that has been cut in one place to produce a linear version of this plasmid

46
Q

What are the steps for loading the gel in gel electrophoresis?

A
  1. The plasmid DNA sample is mixed with 5 ul of loading buffer. The loading buffer contains glycerol to ensure that the DNA samples sink in to the wells in the agarose gel, and bromophenol blue, which acts as a marker dye to indicate the progress of electrophoresis
  2. Using a pipette, 15 ul of the prep./loading buffer mix can be loaded into one of the lanes of the gel
  3. The samples are electrophoresed at 80-100 V until the bromophenol blue has migrated to within 2 cm of the end of the gel. This takes about 45 min – 1 hour.
  4. The DNA can be visualised under UV light using a UV transilluminator and photographed
47
Q

If chromosomal DNA has contaminated the plasmid DNA preparation where will it be seen?

A

Chromosomal DNA is much larger than plasmid DNA, and if present will be seen running higher in the gel than the 10 kbp marker fragment (it may run as a smear if it has been sheared or degraded during the prep)

48
Q

In what order will you see the supercoiled, linear and relaxed DNA in the gel electrophoresis?

A
  • The supercoiled DNA will run the fastest and will be seen nearer the bottom (corresponding to the smaller marker)
  • the linear DNA runs the second fastest and will usually be seen in the middle
  • the relaxed DNA will run the slowest corresponding with the larger DNA markers and probably seen nearer to the top
49
Q

Where will contaminating RNA be seen in the gel electrophoresis?

A

It will run ahead of all the markers a s a ‘cloud’ or bright smear. This bright smear may also contain smaller pieces of sheared DNA

Introduction to Laboratory Science (5 decks)

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