Download Isolation of plasmid DNA

Notes for Preparation/Demonstration of Practical Session:
Isolation of plasmid DNA and quantification using UV spectrophotometry.
Aims and Objective of Practical:
Preparation of plasmid DNA is one of the fundamental techniques used in biotechnological
cloning regimes and this practical session trains students in • underlying chemical principles of classical ‘3 step’ mini prep protocol
• use of basic equipment (centrifuges, pipetting etc. )
• principles of stock dilutions and basic DNA calculations
Note: This session could follow on from Preparation and Transformation of Competent
E.coli and Selection of Transformants and links to both Restriction Digestion and Agarose
Gel Electrophoresis and/or Polymerase Chain Reaction practical sessions and the
Bioinformatics workbook.
Additional documents provided for use:
• Technical support and preparation notes
• Practical demonstrators introductory notes.
Suggested organisation of students: Ideal for small group work/ pairs or for individual
work.
Risk Assessment and Safety Considerations: Requirements and regulations will vary
from country to country and should be checked and the safety information provided for
students modified accordingly before the practical is undertaken. Disposal regulations
should also be checked carefully. A suggested information box for the UK is given on the
front page of the sample schedule.
Learning Outcomes for Students: Suggested at start of schedule.
Assessment:
The practical session can be assessed in several ways, if required, and can be adapted
depending on level of student, prior experience and learning outcomes of the programme
of study in which the practical will be delivered e.g. either via a traditional ‘write up/report’,
with data being collected during the practical session and written up later, or by a mixture
of multiple choice questions and guided calculations etc. Assessment based on
calculations and answers to multichoice questions are given.
A sample student practical schedule is attached. This should be adapted depending on
level of student/ prior experience/ facilities available in lab etc. Many protocols are
available for use, the one given being adapted from Sambrook J. Fritsch E.F. and T.
Maniatis.(1989). Molecular Cloning A Laboratory Manual. Second edition. Cold Spring
Harbor Laboratory Press
Time Required: Session fits in 3 hour slot.
Technical support and preparation notes
All plastic/ glassware should be autoclaved and dried before use, or purchased presterilised. Water used should be double distilled and deionised. Solutions should be
aliquoted and autoclaved or filter sterilised before use, as indicated.
Advanced preparation:
Bacterial media:
Luria broth (LB): For 1 l: 10 g tryptone, 5 g yeast extract, 10 g NaCl (RMM 58.44), make up
in double distilled and deionised water and pH to 7.0. Autoclave for use. [Small volumes
may be filter sterilised].
Ampicillin, 10mg/ml. Filter sterilise [do not autoclave]. Store at –20oC for up to 1 year. Use
ampicillin at a final concentration of 0.1 mg/ ml (i.e. 1 ml of 10 mg/ ml stock solution per
100ml of media).
Bacterial cultures and pellets: at least 12 hours in advance inoculate LB ampicillin medium
with a single bacterial colony containing the desired plasmid and incubate at 37 0C
overnight on a roller drum or shaking incubator. Pellet 5ml aliquots at >2000g for 10mins
at 4oC shortly before practical session, discard the supernatant and store pellets on ice.
1µg/ml solution of pGLO (or equivalent plasmid DNA) solution. Prepare as outlined in the
schedule below or purchase commercially.
Solutions and Buffers:
GTE solution: 50mM glucose, 10mM EDTA, pH8, 25mM Tris.Cl, pH8.
To make up 100ml (with ddH20):
Stock solution
Volume (ml)
Final concentration
(mM)
40% w/v glucose
2.27
50
0.5M EDTA, pH 8
2
10
1M Tris.Cl, pH 8
2.5
25
o
NB: use sterile stock solutions and store at 4 C
Lysis Solution: 0.2N NaOH, 1% SDS.
To make up 10ml (with ddH20):
Stock solution
Volume (ml)
1N NaOH
2
10% SDS
1
Final concentration
0.2N
1%
NB: Prepare fresh prior to use
3 M Potassium Acetate solution.
To make up 100ml (with ddH20):
Stock solution
Volume (ml)
5 M KOAc
60
glacial acetic acid
11.5
1M Tris.Cl, pH 8
2.5
NB: filter sterilise. The resulting solution is 3 M potassium and 5 M acetate and has a pH of about 4.8
RNAase: 10 mg/ml. Dissolve 100 mg RNAase A (pancreatic RNAase) in 10 ml 10mM,
Tris-HCl 15 mM NaCl. Heat to 100oC for 15 minutes and then allow to cool slowly to room
temperature. Dispense into aliquots and store at -20 degrees C.
TE buffer; 10mM Tris.Cl pH 7.5, 1mMEDTA pH8. Dilute from 1M Tris.Cl, pH7.8 and 0.5M
EDTA, pH8 sterile stocks. For 100ml TE- 1ml 1M Tris Cl, pH7.8 and 0.2ml EDTA, pH8.
Equipment/solutions required for session:
• Microcentrifuge
• Whirlymixer
• Microcentrifuge tubes
• Microcentrifuge rack
• P1000 Gilson micropipettes (or equiv)
• P200 “
“
“
• P20 “
“
“
• gilson micropipette tips
• Solid (tip) waste container
• Liquid waste container
• Stop clock
• Indelible marker pen
• Ice bucket and crushed ice
• 10% bleach v/v bleach
solution or disinfectant (e.g. virkon)
• Pellet of ~5ml overnight culture of E.coli
• TE buffer
• GTE solution
• Lysis solution
• Potassium Acetate solution
• RNAase: 10 mg/ml
• UV spectrophotometer
• Quartz cuvettes
Amount / group
1 (per 2-4 groups, ideally)
1 (per 2-4 groups, ideally)
~10
1
1 (ideally)
1“
1“
box p1000&p200/20 / group
-1 (can share)
-1 (can share)
1
1
1 (can share)
bottle (to share)
1 per group.
5 ml
0.5 ml
0.5 ml
0.5 ml
10µl
1 per session
1 per group (ideally)
Predicted Results:
• As given in the practical schedule:
o Expected yield of DNA 2-10µg
o A260/A280 ratio ~
For additional technical help, recipes and protocols see: Sambrook J. Fritsch E.F. and T.
Maniatis.(1989). Molecular Cloning A Laboratory Manual. Second edition. Cold Spring
Harbor Laboratory Press; Ausubel FM. Brent R. Kingston RE. Moore DD. Seidman JG.
Smith JA. And K Struhl (eds) (1998) Current Protocols in Molecular Biology. John Wiley &
Sons, or equivalent.
Practical demonstrators introductory notes.
Draw student attention to
• the hazards and safety considerations that they have to be aware of (detailed in the risk
assessment box) before they start work
• Lab coats and glasses on at all times;
• Live cells being used- so nothing down sink; liquid and solid waste to be segregated and any
spills disinfected. Note handwashing- at end of practical, and if contaminated with cells.
• Students must read and prepare ahead, note solutions need to be pre-chilled etc.
• Gloves should be worn throughout (in addition to personal protection from hazards) to prevent
contamination of plasticware with nucleases from exposed skin
• Use correct pipettes [p1000 for 200-1000µl; p200 for 20-200µl; p20 for <20µl]. Remind how to
set pipette correctly
• Draw attention to importance of fully resuspending cell pellets
• Draw attention to importance of keeping/ discarding correct phase at different steps
• Outline organisation of session (numbers per group/ number of groups/ location of equipment
etc.)
• Explain assessment. [Suggested] assessment is a series of calculations and questions at the
end of the schedule that should be completed (so, be aware of schedule when organising
benchwork!) Note on copying and plagiarism: everyone works in groups and are welcome to
discuss results, but answers but be an individuals own work!
Run through essence of practical (detailed in schedule, so no need to go into fine points)
• Minipreparation of plasmid DNA using classical 3 step protocol
o 1: alkaline lysis of cells
o 2: neutralisation and precipitation of cellular debris and chromosomal DNA with
acetate and SDS
o 3: precipitation of plasmid DNA with isolpropanon
•
Quantification of isolated DNA using A260. Determination of purity determined from
A260/A280 ratio.
Expected results.
As outlined in schedule:
i) The expected yield of DNA from a 5 ml culture are 2-10 ug.
ii) DNA has a A260/A280 ratio of pure DNA = 1.8. Often lower- as a result of contamination
with protein.
ISOLATION OF PLASMID DNA AND QUANTIFICATION USING UV
SPECTROPHOTOMETRY
COSHH Risk Assessment and Safety Considerations
Read the following and sign below before you begin your practical work:
Sodium dodecyl sulphate (SDS). Is toxic and an irritant. Wear gloves
and a lab coat and if spilled on the skin rinse with plenty of water.
Sodium hydroxide is highly corrosive. Wear gloves and a lab coat and
if spilled on the skin rinse with plenty of water.
Isopropanol is flammable, corrosive and carcinogenic. Do not use it
near a naked flame. Wear gloves and a lab coat and if spilled on the
skin rinse with plenty of water.
Microcentrifuges rotate at very high speeds. Always ensure that tubes
are positioned in a balanced configuration and that all lids (inner covers
are provided for some instruments) are closed tightly.
Escherichia coli: ACDP category 1 (minimum risk). Dispose of live
cells and anything that has been in contact with them in the designated
waste containers for autoclaving. Wash hands thoroughly after
handling, or if contaminated, with anti- microbial detergent. Wipe up
any spills with disinfectant.
Signature: ___________________________
Learning Outcomes:
At the end of the practical session and directed learning you should be able to:
Isolate plasmid DNA using a standard practical procedure
Calculate the amount of DNA isolated using UV spectrophotometry and
comment on the purity of the DNA isolated
Discuss the relevance of each main step in the practical procedure and the
importance of the ability to isolate DNA in molecular biotechnology
INTRODUCTION
The ability to isolate good quality plasmid DNA is a fundamental requirement of molecular
biotechnology and gene cloning applications. This practical allows you to gain experience
of one of many chemical methods used to isolate and purify an antibiotic resistance
plasmid from a transformed strain of E. coli (see associated practical Preparation and
transformation of competent E.coli and selection of transformants).
Many laboratories now routinely isolate plasmid DNA using a number of relatively cheap
and reliable commercially available kits, however it is valuable to understand the principles
underlying isolation which are illustrated in the following classical ‘3 step’ practical
protocol: a strain of antibiotic sensitive E. coli transformed with an antibiotic resistance
plasmid are grown in the appropriate antibiotic (e.g. ampicillin for pGLO) (1) The cells are
lysed with alkali. (2) The cell debris and chromosomal DNA is precipitated with SDS and
potassium acetate. After pelleting the debris (3) the plasmid DNA is precipitated from the
supernatant with isopropanol and the DNA resuspended in TE buffer.
The procedure given will usually yield 2-10µg DNA, i.e. it is a small scale, or
minipreparation of DNA, however it can be scaled up many fold to give midi or maxi
preparations, which will yield up to 1 mg DNA from a 1 l starting culture, depending on how
much DNA is required for downstream manipulations.
The isolated DNA is easily quantified by UV spectrophotometry at 260nm. A pure doublestranded DNA solution at a concentration of 50 g/ml will have a spectrophotometric
absorbance A260 (or optical density OD260) of 1.0 at a wavelength of 260nm. In addition, if
the absorbance at 260nm is also measured the relative purity of the isolated DNA can be
determined, given that pure DNA has a A260/A280 (OD260/OD280) ratio of 1.8.
PREPARATION FOR PRACTICAL: please note
(i)
The practical schedule is an involved procedure and you should read ahead and
prepare in advance. For example, solutions have to be pre-chilled etc
(ii)
Make sure that you retain the correct phase at each step.
(iii) Remember that you are initially handling live cells- nothing that has come into
contact with the cells should go down the sink. [if you do forget, report it to a
demonstrator so they can disinfect the sink]
MATERIALS:
Due to the time constraints of the practical you are provided with a cell pellet from a 5ml
overnight culture of E. coli previously transformed with plasmid DNA (see associated
practical Preparation and transformation of competent E.coli and selection of
transformants). Solutions and buffers used in practical: TE: 10mM Tris.Cl, pH7.5, 1mM
EDTA, pH8; GTE solution: 50mM glucose, 10mM EDTA, pH8, 25mM Tris.Cl, pH8; Lysis
Solution: 0.2N NaOH, 1% SDS; RNAase: 10 mg/ml in TE.
METHODS:
A) Isolation of DNA
1. Fully resuspend the bacterial cell pellet in 1 ml of TE buffer and transfer the suspension
to a labelled microcentrifuge tube.
2. Centrifuge for 1 min. at 12 000 g (full speed) in the benchtop microfuge (make sure the
tubes are in a balanced configuration) to pellet the bacterial cells. Discard the
supernatant (carefully pour off all of the liquid, so that the remaining pellet is almost
dry).
3. Resuspend each pellet in 200 µl GTE solution, transfer the solution to a labelled
microcentrifuge tube and incubate at room temperature for 5 minutes.
Add 400 µl of freshly prepared LYSIS solution and mix gently by inverting the tube
several times (do not vortex mix or shake). Incubate on ice for 5 minutes. [NB: The cells
will rapidly lyse in the alkaline buffer and as they do so, you should observe the cell
suspension clearing; if you open the microcentrifuge tube you should note the lysed
solution has become ‘snotty’].
5. Add 300 µl of an ice-cold solution of 3 M POTASSIUM ACETATE solution. Mix gently
by inversion and incubate on ice for 5 min [NB: The potassium acetate neutralises the
NaOH in the lysis buffer and at this stage you should see a thick white precipitate of cell
debris and chromosomal DNA forming]
6. Centrifuge for 1 min at top speed in the microfuge and transfer the supernatant to a new
labelled tube (be careful not to transfer any of the precipitate- if you do then repeat the
centifugation).
7. Add 0.6 X the sample volume (540 µl) of ice cold isopropanol, mix by inversion and
incubate at room temperature for 2 min (NB: at this step the DNA should precipitate out
of solution, which might make the solution slightly turbid, however do not worry if there
is little visible turbidity- as you are only dealing with microgram quantities).
Pellet the nucleic acid by centrifugation for 1 min at top speed in a microfuge and
discard the supernatant [NB the DNA pellet might not be easily visible- however if you
undertake the centrifugation with the hinge of the microcentrifuge tubes facing outwards
then you will know that the pellet will be directly underneath it).
Wash the pellet twice with 1 ml ice cold 70% v/v ethanol, by adding the ethanol, vortex
mixing briefly, centrifuging for 30 sec and removing the ethanol with a micropipettor
(NB: take care not to discard the pellet at this step, which might become ‘whiter’ and
more visible).
Spin for 1 min and discard the supernatant. Air dry the pellet for 5-10 min (there should
be no odour of ethanol remaining once the sample is dry).and resuspend in 50 µl TE pH
7.5. Add 1 µl of a 10 mg/ml solution of RNAase and incubate for 15- 30 minutes at room
temperature.
B) Quantification of DNA by UV spectrophotometry
Using TE buffer as the diluent, make an appropriate dilution of your DNA depending on the
size of the cuvettes available (e.g. for 3ml cuvettes, dilute 30l DNA solution in 2970l of
TE). Determine the absorbance at 260 nm AND at 280 nm, using TE as the reference
solution (i.e. as a blank)
Note:
Calculation of yield of plasmid DNA is given by: The A260 of DNA at a concentration of 50
g/ml is 1.0. NB. The expected yields from a 5 ml culture are 2-10 ug.
The purity of DNA is indicated by: The A260/A280 ratio of pure DNA is 1.8.
NB. if contaminated with protein, the ratio is usually reduced.
Results:
Wavelength (nm)
260
280
Absorbance
Assessment:
1.
Calculate the concentration of your plasmid DNA solution [remember to take into
account the dilution made] and comment on your yield.
2.
Calculate the A260/A280 ratio of your DNA solution and comment on its purity.
3.
Discuss 3 applications of the DNA you have isolated.
4.
A
Why is ribonuclease included at step 10?
To digest ribonucleic acid so the extracted plasmid DNA is free of contaminating
nucleotides
To digest proteins which keep the plasmid and chromosome DNA together
To make the chromosomal DNA precipitate
To digest DNA so the extracted plasmid ribonucleic acid is free of contaminating
nucleotides
To digest the bacterial chromosome DNA
B
C
D
E
5.
Lysis solution contains 0.2N NaOH. The formula weight (molecular weight) of NaOH
is 40. If you were preparing 100 ml of this buffer, what mass of NaOH would you
need?
A
C
E
6.
A
B
C
D
none of the amounts below
B
0.4 g
0.8 g
D
4.0 g
8.0 g
Which of the following is true regarding plasmids?
"Plasmid" is another name for the bacterial chromosome
Plasmids are found in the nucleus
Plasmids contain only "junk" or "spacer" DNA
Genes in plasmids code for proteins with specialised functions, such as antibiotic
resistance
Plasmids carry out protein synthesis using ribonucleic acid (RNA) as template
E
Answers.
1. A260 reading x 50 x dilution factor µg/ml. Units should be adjusted if required. Plus
concise and clear comment on yield gained as opposed to that expected.
2. A260 / A280 ratio given. Plus concise and clear comment on purity gained as opposed to
that expected.
3. A whole host of possible answers could be given.
4. A
5. C
6. D