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Useful Information:
Molecular Biology WorkShop, Use the notes that you have taken in the lectures and from your textbooks to add to the information here. Remember, these laboratory classes illustrate the theory taught in the lectures and as such the notes you make here can also be used to help you revise
Agarose gel electrophoresis is used to separate DNA fragments of different sizes.
Agarose is a non-sulphated linear polymer consisting of alternating residues of D-galactose and 3,6-anhydro-L-galactose. Gelling properties are attributed to both inter- and intra-molecular hydrogen bonding. Due to their hydrophilic nature and the nearly complete absence of charged groups, agarose gels cause very little denaturation and adsorption of sensitive biological substances. Agar consists of two main components: agarose (about 70%) and agaropectin – in other words, agarose is very pure agar. Agarose gels are used extensively in molecular biology for the separation of DNA and RNA molecules by electrophoresis.
DNA is negatively charged therefore when an electric current is applied it will move towards the positive electrode, small fragments of DNA moving further than large fragments. A buffer is used to make the agarose gel and to put in the gel tank which allows the current to pass through the gel.
The DNA (and RNA) in an agarose gel needs to be made visible.
• ETHIDIUM BROMIDE (EtBr) is routinely used to stain DNA, Molecular Biology WorkShop resulting in a material that is clearly visible under ULTRAVIOLET IRRADIATION after EtBr staining, so long as sufficient DNA is present. EtBr INTERCALATES between adjacent base-pairs of DNA. It binds specifically to dsDNA (double-stranded), or base-paired regions of ssDNA, Molecular Biology WorkShop with no apparent preference for particular base sequences. This causes partial unwinding of the double helix and different forms of DNA unwind to different extents – a property that is exploited in purification of plasmid DNA.
WARNING: Ethidium Bromide is toxic and therefore gels must be handled wearing gloves.
Other stains can be used which are less hazardous and allow the gel to be looked at with normal light but generally more DNA is required.
• GelRedTM, which we will be using in these labs, Molecular Biology WorkShop is more sensitive than Ethidium Bromide in many situations and is also non-mutagenic and not cytotoxic. You should still, however, follow good laboratory practice at all times.
Sample preparation
A loading buffer must be added to the samples before running a gel; this contains glycerol to make the sample heavy so it stays in the wells in the gel and also a dye so it can be seen. Read the instructions for each gel carefully to ensure that you sue the correct loading buffer and the load the correct amount.
You will also run some DNA marker on the gel. This is DNA which has been cut with enzymes to produce fragments of DNA of known size. Read the schedule carefully so that you know which marker you are using, the size of the fragments and how much to load on the gel.
Agarose Gel Electrophoresis Procedure
Read the schedule to check that you are using the correct buffer and loading he correct volume of each of your samples
1. The agarose gel has had GelRedTM added but you will need to pour it into the tank and allow to set for 15mins. Make sure the end plates and comb are in the slots in the minigel apparatus as shown by the demonstrator.
2. When the gel is set, add buffer and gently remove the end plates, and the comb.
3. Place the black plastic under the tank and you will see a series of wells in which you will put your samples.
4. Load your samples onto the gel following the instructions in the schedule. Ensure that you use a clean yellow tip each time.
Plasmids
Plasmids occur in both Gram negative and Gram positive bacteria and are between 1.5kb and 300kb in size. They are not part of the bacterial genomic DNA but are extra DNA fragments (cccDNA) which give the bacteria phenotypic characteristics such as antibiotic resistance, toxin production or the ability to use certain nutrients.
The two plasmids you are going to use in these labs are plasmid pUC19 and pBM103. The former is 2686 bp in length. The latter is an E.coli cloning vector and carries antibiotic resistance to ampicillin and tetracycline.
The plasmid pBM103 has been inserted into E.Coli HB101 by transforming ‘competent’ cells with plasmid DNA. Competent cells are bacteria that have been treated with magnesium chloride and calcium chloride; Molecular Biology WorkShop this enables them to take up plasmid DNA when subjected to cold and heat shock. The bacteria containing the plasmid can be selected by growing on media containing antibiotics, in this case ampicillin and tetracycline. Only bacteria containing the plasmid will be able to grow. When grown in liquid culture the plasmid will replicate within the bacterial cell.
Plasmids are extracted using a procedure in which the bacteria are lysed (broken open) and the plasmid DNA separated from the bacterial chromosomal DNA.
In these labs you will extract plasmid DNA using two different, but related techniques:-
1. Alkaline lysis miniprep of plasmid DNA
This is the most common method for plasmid minipreps, and a number of commercially available kits are based on this method.
DNA denatures (i.e. separates into single strands) under alkaline conditions. When the pH is returned to neutral the strands attempt to re-anneal. Because of the natural supercoiling of plasmids the denatured strands are unable to move away from each other and thus re-anneal correctly to reform the plasmid. Bacterial chromosomal DNA on the other hand does not re-anneal correctly, resulting in a tangled mass of DNA.
2. A commercial kit method for the extraction of plasmid DNA.
You should consider
• what contaminants you may have in your final plasmid DNA sample
• the yield that you achieve
• the state of your plasmid. Is it linear , supercoiled or circular ?
• which of the two methods that you are using is best
Restriction Endonucleases
These are enzymes (of which there are hundreds) which can be used to cut DNA into small fragments. Each enzyme recognises a sequence of bases 4-16bp long in the DNA and cuts the DNA between specific bases at that point.
Plasmids which are usually double stranded, supercoiled and circular may be cut only once by an enzyme generating a linear piece of DNA or may be cut several places by a single enzyme generating lots of fragments of DNA. These can be separated by gel electrophoresis and by using a number of enzymes a ‘restriction map’ of the plasmid can be constructed. This is useful for identifying and characterising plasmids and planning manipulations with DNA.
The restriction enzymes being used in the second laboratory class all have 1 restriction site in the plasmid being digested so when used singly in a reaction will cut in the plasmid once generating 1 linear fragment. But when used together will generate multiple fragments of different sizes allowing a restriction map to be constructed. The enzymes are EcoR1, Sal 1, PVU11 and Pst1.
In a restriction reaction there needs to be the enzyme, Molecular Biology WorkShop a buffer which provides the right conditions for the enzyme, Molecular Biology WorkShop DNA and water to make a standard volume, usually 15 -20 µl, this is the reaction master mix
The reaction then has to be incubated at 37°C for a set time. Read the schedule for more details.
Spectrophotometric analysis of plasmid DNA
The Nanodrop spectrophotometer will provide a spectrum of your plasmid extract from a very small (generally ~10 μl) sample, as shown below. However, contaminants in the preparation will distort the spectrum. Common contaminants include proteins, Molecular Biology WorkShop which absorb maximally at 280nm, and phenol (absorbs at 270nm). Additionally, Molecular Biology WorkShop Molecular Biology WorkShop organic molecules containing double bonds will absorb at 230nm.
For reasonably pure nucleic acid preparations the A260 measurement can be used to estimate the quantity of DNA or RNA. For DNA, a 50g/ml solution gives an A260 of 1.0 ONLY IF THE A260:A280 is correct. The machine will report your DNA concentration in ng/ μl (convert this to μg/ml – it’s much easier than you think!). IT WILL ASSUME THAT YOUR DNA IS PURE AND CALCULATE ON THE BASIS OF 1 Absorbance unit = 50 μg/ml. This may not be justified if the A260:A280 is <1.8.
A typical Nanodrop spectrum of DNA .
DNA Marker band sizes:
DNA Marker
Fermentas
Fast Ruler Middle Range
NOTES
LABORATORY SESSION (1)
REMEMBER TO READ THE SAFETY INSTRUCTIONS BEFORE YOU START WORK
Each person will perform the plasmid DNA extraction as an individual
Order of tasks for this practical session
• Pour agarose gel and leave to set.
• Isolation of plasmid DNA
• Load agarose gel with the extracted plasmid and DNA markers.
• Spectrophotometric analysis of isolated plasmid DNA (while gel is running).
• Examine and record gel results; plot the calibration curve BEFORE Lab 6
• Make sure you have all the results recorded in your schedule
• You will need to have completed the worksheet questions (part of this lab schedule) BEFORE Workshop 7.
EXPT. 1: Alkaline lysis miniprep of plasmid DNA
In this experiment you will isolate the plasmid pUC19 which is grown in the host bacterium E. coli JM103.
Method Important – Label tubes with your initials so you know which is yours.
1. You are provided with 1.5 ml of E.coli culture containing the plasmid pUC19.
2. Microcentrifuge the tube for 1 min at full speed to pellet the bacteria.
3. Remove the supernatant using an automatic pipette and discard it into the disinfectant beaker. Resuspend the bacterial pellet in 100l GTE solution by pipetting up and down using a 1ml automatic pipette with a blue tip. Allow to sit at room temperature for 5 min.
4. Add 200l NaOH/SDS solution. Mix by capping the tube tightly and inverting it several times. The contents of the tube should become clear and viscous. Place on ice for 5 min.
5. Add 150l potassium acetate (KAc)solution, vortex for 2 sec, then place on ice for 5 min. A white precipitate should be visible.
6. Microcentrifuge for 3 min at full speed.
7. Transfer 400l of the supernatant to a new tube. Avoid transferring any of the white precipitate. Add 800l of 95% ethanol to this supernatant, mix and leave at room temperature for 2 min.
8. Microcentrifuge for 3 min at full speed.
9. Remove and discard the supernatant. Add 1ml of 70% ethanol to the pellet.
10. Microcentrifuge for 1 min at full speed. Remove and discard the supernatant.
11. Remove as much ethanol as possible by inverting the tube onto a piece of tissue. You may gently wipe a clean piece of tissue around the inside top edges of the tube.
12. Leave the tube open at room temperature for 5-10 min to allow the pellet to air dry.
13. Resuspend the pellet in 100l water, by pipetting up and down using a automatic pipette.
This is your bacterial plasmid DNA preparation. You will now analyse this by
1) Spectrophotometry with the Nanodrop – Remove 10l into a separate Eppendorf tube and keep until you have loaded the gel (see EXPT 3, below)
2) Agarose gel electrophoresis – Add 20l of blue loading buffer (this contains bromophenol blue and glycerol) to the remaining 90l of plasmid extract and mix gently, This is to load onto the gel (see EXP 2 below)
EXPT. 2: Analysis of plasmid DNA by agarose gel electrophoresis
IN GROUPS OF 2 STUDENTS, POUR AN AGAROSE GEL.
1. Preparation of agarose gels
a) Make sure the end plates and comb are in the slots in the minigel apparatus as shown by the demonstrator.
b) Pour the 35ml molten 0.8% agarose containing GelRedTM into gel tank and leave the gel to set on a level bench for at least 15min.
c) When the gel is set, add 50ml TBE buffer and gently remove the end plates, and the comb.
d) Place the gel on a sheet of black plastic so you can see the 12 wells.
2. Prepare your samples
a) You have already prepared the plasmid sample for electrophoresis (by adding 20l of blue loading buffer to 90l of plasmid sample in an Eppendorf tube).
3. Load the gel:
a) Arrange the gel in a convenient position close to the power pack. You will not be able to move the gel once you have loaded the samples.
b) Load the samples and markers according to the plan below using an automatic pipette. To load the gel place the pipette tip just below the surface of the buffer, just into the well to be loaded and slowly eject the sample. Do not poke the tip to the bottom of the well. The sample will fall through the buffer and into the well. DO NOT MOVE THE GEL!
c) Connect the gel to the power pack, and run at 60mA per gel for approx 30-45 min. Check your gel is running by observing bubbles in the buffer near the electrodes.
d) At the end of the run turn off the power pack. Wearing disposable gloves bring the gel tank to the gel documentation system, pour off the gel buffer down the sink, taking care not to lose the gel. Only the person handling the gel needs to wear gloves
e) Examine the gels using the gel documentation apparatus, and print an image of each. You will be helped to do this by a demonstrator.
EXPT 3: Spectrophotometric analysis of plasmid DNA
The Nanodrop spectrophotometer will provide a spectrum of your plasmid extract from the 10 μl retained above. Follow the instructions at the beginning of the schedule.
Results and Interpretation
Complete the following questions before workshop 7.
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How would you use the calibration curve to estimate the size of bands observed in your plasmid DNA preparation?
How could you determine of you have extracted supercoiled plasmid DNA?
Attach the nanodrop spectrum here:-
Calculate the concentration of DNA in your extract, in g/l and g/ml.
NOTES:
LABORATORY SESSION (2)
REMEMBER TO READ THE SAFETY INSTRUCTIONS BEFORE YOU START WORK
Order of tasks for this practical session
1. Pour agarose gel and leave to set.
2. Isolation of plasmid DNA
3. Spectrophotometric analysis of isolated plasmid DNA
4. Set off the restriction endonuclease digestions.
5. Load agarose gel with the plasmid digests and DNA markers.
6. Examine and record gel results;
7. Make sure you have all the results recorded in your schedule
• You will need to have plotted the calibration curve and completed the worksheet questions (part of this lab schedule) BEFORE Workshop 7.
EXPT. 4: Extraction of plasmid DNA using a commercial kit.
Working as pairs. Before you start you will need to put on some gloves.
You have been given a tube with E.coli pBM103 culture. Label the lid of the tube with your name.
1. Add 250µl of P1 lysis buffer and resuspend the pellet by gently pipetting up and down until there are no clumps.
2. Add 250µl of P2 buffer and mix by inverting the tube a few times, (the solution should turn blue, this indicates the cells have been lysed)
3. Add 350µl of N3 neutralisation buffer and mix by inverting the tube a few times, (the solution should turn colourless, if it is still blue continue mixing)
4. Centrifuge for 10 mins
You can pour your electrophoresis gel whilst you are waiting.
5. Transfer with a pipette approx 700µl of supernatant to the column in the blue tube.
6. Centrifuge for 1 min then discard the liquid from the tube. The plasmid DNA is stuck to the column
7. Add 500µl of PB wash buffer to the column, centrifuge for 1 min then discard the liquid from the tube.
8. Add 750µl of PE wash buffer to the column, centrifuge for 1 min then discard the liquid from the tube.
9. Centrifuge again for 1 min. (this ensures all the wash buffer is removed) then transfer the column to a new tube.
10. Add 100µl water to the column and leave for 1 min, then centrifuge for 1 min. The plasmid DNA will be released from the column into the water, now discard the column.
This is the plasmid DNA which you are now going to analyse:-
(1) By nanodrop as you carried out in the first laboratory class
You will use a 2µl of sample to measure the absorbance of your plasmid DNA. You will need a [DNA] of at least 15 ng/l. If your plasmid sample is less concentrated you will need to use a pre-prepared sample. See a demonstrator for this.
(2) By restriction endonuclease digestion followed by running the digests on an agarose gel.
EXPT 5: Spectrophotometric analysis of plasmid DNA
Attach the nanodrop spectrum picture here:-
What concentration is your plasmid DNA?
CONSIDER:- Do you have a pure plasmid DNA? Compare the extraction method used this week with the one used in the first lab. Which is better? Explain your decision.
EXPT. 6: Restriction Digest of pBM103
Restriction procedure
You have a set of tubes labelled 1-6 which contain 15µl restriction master mix containing different enzymes as follows:
1 EcoR1
2 EcoR1 + Sal 1
3 EcoR1 + Pvu 11
4 EcoR1 + Pst1
5 EcoR1 + Sal 1 + Pvu 11
6 EcoR1 + Pvu 11 + Pst1
• Add 10µl of your plasmid DNA to each tube and close the lid tightly. You need a [DNA] of at least 15 ng/l. If your plasmid sample is less concentrated you will need to use a pre-prepared sample. See a demonstrator for this.
• Mix gently and centrifuge for 1 min.
• Place in the water bath or heating block at 37°C for 10mins.
Your samples are now ready for analysis by gel electrophoresis
Sample preparation
1. Add 5µl of the blue loading buffer to each of your sample tubes.
2. Mix by tapping the tube and allow the sample to run to the bottom of the tube
3. You are now ready to load the samples onto the gel
You will also run some DNA marker on the gel. This is DNA which has been cut with enzymes to produce fragments of DNA of known size…
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