Introduction to the Molecular Biology Laboratory
Drs. Karen Held Hales and Buck Hales
PHYB 569 Methods in Experimental Physiology
September 19-20, 2000
Overview: the approach of molecular biology vs. biochemistry
Working with bacteria
Plasmids, restriction enzymes, antibiotic resistance
Plasmid preps
Purifying DNA
Analyzing DNA: sequencing, restriction mapping, Southern blot, PCR
Applications for bacterially expressed plasmid DNA
Cloning genes—cDNA vs. genomic DNA
Expressing protein
Transfecting DNA for expression studies and promoter-reporter assays
Stably transfecting DNA to produce new cell lines
Setting up your bench
Solution protocols
Creating a nuclease-free work environment
Agarose vs. acrylamide gels
Working with ethidium bromide
Use of radioisotopes, 32P-dNTPs for labeling reactions
Phenol-chloroform extraction and ethanol precipitation of nucleic acids
Lab component:
Quantitated DNA—purified plasmid DNA, mini-prep purified DNA
Restriction enzyme digest of DNA
Streak plates, inoculate overnights
Analyze restriction digest by agarose gel electrophoresis
Mini-alkaline lysis of overnights
Stock Buffers and working solutions
Stock buffer and working solution formulae are for 100 ml, unless noted.
Stock Buffers:
Use GIBCO type media bottles and autoclave.
*1 M Tris, pH 7.2, 7.4 and 8.0
*0.5 M EDTA, pH 8.0
5 M NaCL [58.6 g, QS 200 ml]
0.75 M Na Citrate, pH 7.0 [22.1 g/95 ml; pH and QS 100 ml]
10 % Na Lauroyl Sarcosine [10 g QS 100 ml]
10% SDS [10 g QS 100 ml]
*see below
Basic Working Solutions:
Use GIBCO type media bottles and autoclave.
*TE pH 7.2 (for RNA)
*TE pH 7.4 and TE pH 8.0 (for DNA)
4 M ammonium acetate (NH4OAc) [30.8 g, QS 100 ml]
10 M NH4OAc [77 g, QS 100 ml]
3 M sodium acetate, pH 5.6 (NaOAc) [24.6 g/75 ml, pH with HCL, QS 100 ml]
2 M NaOAc, pH 4.0 [16.8 g in 50 ml glacial HOAc, QS and pH with H 2O]
PBS, pH 7.1 (prepare from 10X, adjust pH, autoclave) store at 4 C
"RNA-H2O" (autoclave MQ H2O in presterilized 100 and 500 ml bottles)
1M Tris stocks (100 ml each)
12.11 g Tris base, dissolved in 70 ml H 2O.
adjust to the appropriate pH with conc. HCl:
pH 7.2, add conc. HCl, approx 8-10 ml, the QS to 100 ml
pH 7.4, add conc. HCl, approx 6-8 ml, then QS to 100 ml
pH 8.0, add conc. HCl, approx 2-5 ml, then QS to 100 ml
0.5 M EDTA (100 ml)
18.6 g (Na)2-EDTA, dissolve in 90 ml H2O.
Adjust pH of H2O to > 8 by adding several solid NaOH pellets, then add
the EDTA and stir until solution is clear. If pH goes lower than 8, add
more NaOH pellets. After EDTA is in solution, QS 100 ml.
*TE = Tris-EDTA which is prepared from Tris (1M) and EDTA (0.5 M) stocks, and
designated by the pH of the Tris stock.
1 M Tris
1.0 ml
0.5 M EDTA
0.2 ml
H2O
90 ml
adjust pH (if necessary) then QS 100ml
10 X PBS
1 liter
11.4 g
monobasic sodium phosphate
57.5 g
dibasic sodium phosphate
87.6 g
NaCl
dissolve in 750 ml H2O then increase volume to 950 ml. Adjust to pH 7.4 with
either H3PO4,or NaOH, QS 1000 ml. Store in 100 ml aliquots at -20 C.
20X SSC (1X = 0.15 M NaCl/ 0.015 M Na citrate, pH 7.0)
4 liters:
701.4 g NaCl
352.9 g Na Citrate
dissolve in 3000 ml MQ H2O, adjust to pH 7.0 with dilute HCl (it's a weak
buffer), QS 4000 ml.
Tris-borate-EDTA buffer (TBE) for agarose gels:
4 liters of 5X (dilute to 20 liters and store in carboy)
218.1 g Tris base
19.0 g Na2EDTA
111.3 g Boric acid
Loading dye for DNA gels:
BPB/XC (mixed 50:50)
XC: xylene cylanol saturated 50% glycerol, diluted 1:4 with 50% glycerol
BPB: bromophenol blue saturated 50% glycerol, diluted 1:4 with 50% glycerol
10X MOPS: (200 mM MOPS/ 50 mM Na acetate/ 10 mM EDTA, pH 7.0)
1 liter:
41.86 g MOPS
4.1 g Na acetate
3.72 g EDTA
dissolve in 900 ml MQ H2O, adjust pH to 7.0 with NaOH, QS to 1 L. Store
in sterile, brown glass or foil covered 500 ml bottles
Solution D stock buffer for RNA extraction:(4 M Guanidine thiocyanate, 25 mM
Na citrate, pH 7.0, 0.5 % Na sarcosyl)
250 g GTC dissolved in 293 ml H2O at 65o
add 17.6 ml 0.75 M Na Citrate, pH 7.0
add 26.4 ml 10% (w/v) Na sarcosyl
sterile filter
(store at RT in sterile bottle 3-4 months)
Solution D+ working solution:
add 0.36 ml b-mercaptoethanol to 50 ml D
(store at RT for 1 month)
Running buffer for agarose-formaldehyde gels :
1 liter
100 ml 10X MOPS, 87 ml formaldehyde, 813 ml H2O.
Phenol-Chloroform Extraction and ethanol precipitation of nucleic acids
Phenol-Chloroform Extraction:
Materials:
Water or buffer* saturated ultra-pure phenol (4 C)
24:1 chloroform:isoamyl alcohol (4 C)
*water-saturated phenol is less prone to oxidation and can be stored for longer
Method:
1)
2)
3)
4)
5)
6)
Mix DNA solution:phenol:chloroform at a 1:1:1 ratio, vortex 2-5 seconds
Centrifuge full-speed for 2-3 min at RT in Eppendorf type centrifuge
Careful remove upper (aqueous) layer and transfer to new tube
Mix DNA solution (aqueous) 1:1 with chloroform, vortex 1-2 seconds
Repeat steps 2 and 3
Proceed to ethanol precipitation
Ethanol Precipitation:
Materials:
95% ethanol (–20 C)
70% ethanol (-20 C)
Salt solution:
To precipitate nucleic acids from an aqueous solution, increase the salt concentration,
add cold ethanol and place in the freezer. Three salts are generally used for ethanol
precipitations.
salt
NH4OAc
NH4OAc
NaOAc
NaCl
conc. of stock
10 M
4M
3M
5M
final conc. to ppt
2-2.5 M
2-2.5 M
0.3 M
0.15 M
It is often most convenient to use a volume of 4 M NH4OAc equal to the volume of the
aqueous solution of nucleic acid you are precipitating.
To precipitate nucleic acids samples (RNA or DNA) for gel analysis, it is important to
dilute them to the same volume, then add an equal amount of salt to each sample, then
add ethanol. Add 2 to 3 volumes of -20 C ethanol to the salt plus nucleic acid solution,
then place the samples in the freezer.
The following illustrates a precipitation using NaOAc.
a)
To 100 l of DNA solution, add 12 l 3 M NaOAc (final concentration = 0.3 M)
and 225 l 95% ethanol (2 volumes). Precipitate at -20 C for >1 h or
-80 C for 15-20 min.
b)
Centrifuge full speed in microfuge at 4 C for 15 min, decant supernatant.
c)
Wash with 100 l 70% ethanol, centrifuge full speed in microfuge at RT for 2-3
min, decant supernatant.
d)
Lyophilize in speed-vac for 5-10 min until no trace of liquid remains (or air dry on
bench top or in dessicator).
The volume of the sample determines which salt is used for precipitation. Keep the final
volume (sample plus salt plus ethanol) < 1.5 ml to allow all procedures to be conducted
in eppendorf tubes.
LB agar and growth medium
LB:
1 liter
Bactotryptone
Yeast extract
NaCl
10 g
5g
10 g
Dissolve in 900 ml H2O, adjust to pH 7.0 with 4 M NaOH, QS 1 liter
Autoclave.
500 ml
Bactotryptone
Yeast extract
NaCl
5g
2.5 g
5g
Dissolve in 450 ml H2O, adjust to pH 7.0 with 4 M NaOH, QS 500 liter
Autoclave.
Let cool to 50 C, add antibiotics (as per agar plates) and inoculate. Note that
you can increase antibiotic concentration for more stringent selection, up to 2X
concentrations shown below.
For agar plates, usual 500 ml is enough for 20 plates. Prepare as described
above, add 7.5 g Bactoagar/ 500 ml, and autoclave. Let cool to 50 C, add
antibiotics, and pour plates. Then use bunsen burner to quickly sterilize top of
agar and remove bubbles.
For ampicillin plates (AMP)
Prepare amp stock, 50 mg/ml stock, stored at -20 C
Let molten LB agar cool to 50 C, add 1l/ml amp stock, swirl and pour.
Final concentration 50 g/ml.
For tetracyclin plates (TET)
Prepare tet stock, 12.5 mg/ml in 50% ethanol, stored in dark at -20 C
Let molten LB agar cool to 50 C, add 4 l/ml tet stock, swirl and pour.
Final concentration 50 g/ml.
For kanamycin plates (KAN)
Prepare kan stock, 50 mg/ml, stored at –20 C
Let molten LM agar cool to 50 C, add 1 l/ml kan stock, swirl and pour
Final concentration 50 g/ml
In stead of adding antibiotics from stock solutions, you can weigh out powder
and add it directly to cooled but molten LB agar.
Large Scale Plasmid Prep and CsCl Gradient Purification of DNA
MATERIALS:
Ethidium Bromide, 10 mg/ml (EtBr)
NaCl-saturated H2O-saturated isopropanol
TE pH 7.4
TE pH 8.0
LB Broth:
Bacto-Tryptone
10 g
Yeast Extract
5g
NaCl
10 g
Dissolve in 900 ml of H2O, adjust to pH 7.0 with 4 M NaOH, QS 1000 ml, sterilize
by autoclaving.
Solution I:
Solution II:
Solution III:
per 500 ml
50 mM glucose (dextrose)
25 mM tris, pH 8.0
10 mM EDTA
(Sterile Filter)
(prepare fresh)
0.2 N NaOH
1 % SDS
5 M KOAc
Potassium acetate
glacial acetic acid
H2O
(sterilize by autoclaving)
4.5 g
12.5 ml (1M)
10 ml (0.5M)
10 ml 10 N
50 ml 10 %
440 ml H2O
147.3 g
54.5 ml
QS 500 ml
METHOD:
1)
Grow 5-10 ml overnight culture of plasmid-bearing bacteria in LB with antibiotic.
Grow over night or for ~8 hours.
2)
Inoculate 500 ml to 1 L LB plus antibiotics with 5 to10 ml of overnight culture and
shake vigorously overnight at 37 C.
3)
Harvest cells: centrifuge in 500 ml bottles, 2800 rpm in GSK for 30 min at 4 C or
in 250 ml bottles in GSA rotor in Sorval RC-5 5000 rpm for 10 min at 4 C.
4)
Decant supernatant then resuspend each pellet 20 ml of Solution I using a 10 ml
pipet with Pipet-aid. Add an additional 20 ml of Solution I that contains 4 mg/ml
lysozyme (= 80 mg) (prepared fresh) and swirl to mix. Let stand at room temp for
10 min.
5)
Add 60 ml of Solution II (prepared fresh). Mix well by swirling. The solution
should become fairly clear and viscous almost immediately. Let stand at room
temp until clear, approximately 5 min.
6)
Add 60 ml of Solution III. Cap and invert to mix. Mix very well. A fluffy white ppt
will form upon mixing. Allow to stand at room temp about 10 min.
7)
Centrifuge at 8000 rpm for 20 min at 4 C. Use GSA rotor in Sorval centrifuge.
(note, after this spin allow rotor and centrifuge to warm up towards room temp)
8)
Pellet should be white and hard. Pour supernatant through glass wool-plugged
funnel into another 250 ml bottle.
9)
Add 80 ml isopropanol to supernatant and let stand at room temp for 15 min.
10)
Centrifuge at room temp, 8000 rpm for 30 min. Pour off liquid and drain pellet.
11)
Resuspend pellet in 4 ml TE, pH 8; transfer to 15 ml conical tube; add 7.8 g CsCl
and 0.5 ml EtBr and adjust volume to 10 ml TE, pH 8, using graduations on tube.
12)
Centrifuge in clinical type table top centrifuge at full speed for 5 minutes to clarify.
Avoid precipitate or "floating pellet" if one occurs.
13)
Transfer to ultrcentrifuge tubes and balance tubes to within 0.05 g. Use 50 50 %
(w/v) CsCl in TE, pH 8, to balance. To prepare 50% (w/v) CsCl dissolve 30 g
CsCl in 30 ml TE or water. Note: 100 l of CsCl = 0.159 g (Ri = 1.3893).
14)
Centrifuge at 50,000 rpm for ~24 hours at 20 C in Ti80 rotor in ultracentrifuge.
15)
Pull bands from gradient tube with 20 G needle and a 3 or 5 cc syringe. 1st,
insert needle through septum to vent. Wrap tube with scotch tape to seal hole
then insert needle just below band (visible with UV light) with bevel up, and
remove with gentle suction. If you see two bands, remove top band and discard
to avoid contamination (=linear or genomic DNA), then remove plasmid or lower
band.
16)
If preparing DNA for transfections repeat step 14-16 after adjusting volume to 10
ml with 50%(w/v) CsCl.
17)
Transfer to 15 ml conical tube and add 2 volumes of NaCl saturated H2O
saturated isopropanol (top layer). Shake to extract EtBr. Let phases separate
and remove top (pink = EtBr containing) layer. Repeat until pink color is gone
(usually 5 times). Check aqueous layer with UV transilluminator to determine if
EtBr has all be removed.
18)
Adjust volume to 10 ml with H2O. Transfer aqueous phase to 30 ml COREX
TUBE and fill tube with ethanol. Precipitate at -20 C overnight. Note: if a large
and visible precipitate forms immediately, ok to proceed to step 19.
19)
Centrifuge at 9500 rpm for 30 min at 4 C in HB4 swinging bucket rotor in Sorvall.
Pour off supernatant and allow pellet to air dry. Resuspend in TE pH 7.4 (or pH
8.0), add 3 M NaOAc and reprecipitate with 3 volumes of 95% ethanol. Spin,
rinse pellet with 70 % ethanol, and dry in vacuum. Resuspend in 0.5 to 1 ml TE,
pH 7.4 (or pH 8.0).
20)
Quantitate by measuring A260 and determine A260/280 ratio.
(A260)(50)(dil)(volume) = g plasmid DNA
g DNA/volume = concentration g/l
Alkaline lysis mini-prep
1. Pellet cells from 2ml overnight culture in eppendorf tube.
2. Resuspend cell pellet in 100 l solution I containing 100 g/ml RNase A using
vortex.
3. Immediately add 200 l solution II, invert twice.
4. Add 150 l solution III, shake by hand.
5. Centrifuge 1 minute, remove supernatent to new tube and add 900 l ice-cold 95%
ethanol.
6. Mix, centrifuge 1 minute, pour off supernatent and dry pellet.
Note: see full scale plasmid prep for solutions I-III.