DNA Methods

 

General Cloning Methods

Crude Plasmid DNA Preparation by Alkaline Lysis

Solutions: Qiagen Plasmid Preparation Kit

Resuspension buffer (P1): Tris/HCl 50 mM pH 8.0, EDTA 10 mM, RNase A 100 µg/mL , stored at 4°C

Lysis buffer (P2): NaOH 200 mM, 1 % SDS

Neutralization buffer (P3): KAc 3 M pH 5.5

Isopropanol, room temperature

Ethanol 70 %, 4°C

50 mL Falcon tubes filled with 5 mL of LB broth containing appropriate selective antibiotics (ampicillin: 100 µg/mL or kanamycin: 25 µg/mL) are inoculated with one colony of the desired transformed E. coli strain and incubated o/n at 37°C with vigorous shaking at 200 rpm. On the next day, a glycerol stock is made and then the cells are harvested with centrifugation at 6000 rpm? for 15 minutes at 4°C (centrifuge: Heraeus model Biofuge Primo R), the supernatant is removed and collected for autoclaving. Leftovers of LB broth can decrease the yield of plasmid DNA, therefore the Falcon tubes are placed upside down on a paper towel for some minutes in order to remove as much LB broth as possible. The E. coli pellet is resuspended in 250 µL of Resuspension buffer, transferred to 1.5 mL reaction tubes, then 250 µL of Lysis buffer are added and the lysis mix is carefully inverted 5 – 6 times until the solution is viscous. Shearing of genomic DNA by heavy shaking should be avoided. After 5 minutes incubation time at room temperature 250 µL of Neutralization buffer are added and again the mix is carefully inverted several times until a white precipitate of genomic DNA and proteins is visible. The white precipitate is pelleted by centrifuging at 6000 g for 15 minutes at 4°C. The supernatant containing plasmid DNA is collected without perturbing the pellet and centrifuged a second time to quantitatively remove genomic DNA precipitate. The collected supernatant is then subjected to isopropanol precipitation. The unpurified plasmid DNA solution is mixed with 0.7 volumes of isopropanol. After 5 minutes of incubation at room temperature, precipitated plasmid DNA is recovered with centrifugation at full speed, 16 000 x g, at 4°C for 30 minutes (centrifuge: Heraeus Sepatech Megafuge 1.0 R), the supernatant is removed and discarded. The plasmid DNA pellet is washed with 1 mL 70 % EtOH (4°C) without disturbing the pellet and centrifuged for another 5 minutes at full speed at 4°C and the supernatant is quantitatively removed. The pellet is dried for 5 min at RT and dissolved in 20 µl AD or 1x TE buffer. In some cases this DNA-solution was further purified by an EtOH/NaAc precipitation. The crude preparation of plasmid DNA can be subjected to restriction digest analysis and sequencing but cannot be used for transfection experiments of mammalian cells. In the latter case, the crude DNA solution was purified by Qiagen DNA purification columns.

 

Ethanol/NaAc Precipitation of Plasmid DNA

Solutions:

Ethanol 70 %, -20°C

Ethanol 70 %, 4°C

NaAc 3 M

TE buffer: Tris/HCl 10 mM pH 7.5, EDTA 1 mM

The DNA solution or in some cases the restriction digest sample was mixed with 1/10 volume of NaAc, 2.5 volumes of 100 % ethanol (-20°C) and was quickly transferred to -70°C for 5 minutes incubation. Then the plasmid was collected by centrifugation for 30 minutes at full speed, 16 000 x g, 4°C (centrifuge: Heraeus Sepatech Megafuge 1.0 R), the supernatant was removed by suction. The pellet was washed with 1 mL 70 % ethanol (4°C) and again centrifuged for 5 minutes at 14 000 rpm, 4°C and the supernatant was quantitatively removed by suction. The pellet was air-dried for some minutes prior to elution in an appropriate volume of AD or 1x TE buffer (to reach a final concentration of about 1 µg/µl).

 

Plasmid DNA Preparation by Alkaline Lysis:

Solutions:

Equilibration buffer (QBT): NaCl 750 mM, MOPS 50 mM pH 7.0, 15 % isopropanol, 0.15 % Triton X-100, stored at room temperature

Wash buffer (QC): NaCl 1 M, MOPS 50 mM pH 7.0, 15 % isopropanol, stored at room temperature

Elution buffer (QF): NaCl 1.25 M, Tris/HCl pH 8.5, 15 % isopropanol, stored at room temperature

For midi (maxi) DNA preparations the QIAGEN Plasmid Midi (Maxi) kit was used (Cat.no.12143, 12163). A 250 mL (500 mL) shaking flask filled with 50 mL (100 mL) LB broth containing appropriate selective antibiotics (ampicillin 100 µg/mL, kanamycin 25 µg/mL) were inoculated with the desired transformed single colony of E. coli strain and incubated o/n at 37°C with vigorous shaking at 200 rpm. The cells were harvested by centrifugation at 6000 g for 15 minutes at 4°C (centrifuge: Heraeus model Biofuge Primo R), the supernatant was removed and collected for autoclaving. Leftovers of LB broth can decrease the yield of plasmid DNA, therefore the centrifugation buckets were placed upside down on a paper towel for some minutes in order to remove as much LB broth as possible – remnants of LB on the walls of the tubes were removed by suction. The E. coli pellet was resuspended in 4 mL (10 mL) of Resuspension buffer P1, 4 mL (10 mL) of Lysis buffer (P2) were added and the lysis mix was carefully inverted 5 – 6 times. After 5 minutes incubation time at room temperature 4 mL (10 mL) of Neutralization buffer were added, again the mix was carefully inverted several times until a white precipitate of genomic DNA and proteins were visible and the neutralized lysate was incubated on ice for 20 minutes. The white precipitate was pelleted by centrifuging at 6000 g for 15 minutes at 4°C. The supernatant containing plasmid DNA was collected without disturbing the pellet and centrifuged a second time 6000 g for 15 minutes at 4°C. In the mean time a Quiagen tip 100 (500) was equilibrated by applying 4 mL (10 mL) of Equilibration buffer on the matrix of the tip, the buffer entered the resin only by gravity flow. The supernatant of the centrifugation was applied on the columns and entered the column by gravity flow. 2 Washing procedures were performed with 10 mL (30 mL) of Wash buffer respectively. Purified plasmid DNA was eluted with 5 mL (15 mL) Elution buffer. After elution DNA was precipitated with 0.7 volumes isopropanol, pelleted by centrifugation at 14 000 rpm for 30 min at 4°C centrifuge: Sorvall RC-5B rotor SA 600), the pellet was air-dried and eluted in 100 µL (500 µl) AD or 1x TE. DNA yield was quantified with an UV spectrophotometer and the plasmid DNA was diluted to a final concentration of 1 µg/µL.

 

Isopropanol Precipitation of Plasmid DNA

Solutions:

Isopropanol, room temperature

Ethanol 70 %, room temperature

The eluted midi (maxi) plasmid DNA solution was mixed with 0.7 volumes of isopropanol (room temperature) and immediately centrifuged for 30 minutes at full speed, 17 000 x g, 4°C (centrifuge: Heraeus Sepatech Megafuge 1.0 R). The supernatant is carefully removed. The DNA pellet is washed with 2 mL (5 mL) 70 % ethanol (room temperature) without disturbing the pellet and another centrifugation step is carried out for 10 minutes at full speed, 4°C, the supernatant is quantitatively removed by suction. The pellet is air dried and dissolved in 100 µL AD. DNA yield is quantified and the plasmid DNA is diluted to a final working concentration of 1 µg/µL.

 

DNA Quantification

DNA exhibits a specific absorption maximum at 260 nm, which is used for DNA quantification by an UV/VIS spectrophotometer (Pharmacia Biotech Ultrospec 2000). An OD at 260 nm of 1 refers to a DNA amount of 50 µg/mL. An appropriate dilution (1:200 after midi or maxi prep) of the DNA sample is necessary to stay in the linear range (0.1-1) of this quantification method. The purity of DNA concerning the presence of residual proteins can be calculated by the ratio of OD260/OD280. The aromatic amino acid tryptophan exhibits an absorption maximum at 280 nm, which refers to the relative amount of protein in the sample. An OD260/OD280 ratio of 1.8 – 2.0 is desired – which is typical for pure DNA samples resulting from the shoulder of DNA-absorbance at 280 nm.

 

Restriction Digest

Restriction digest of 20 µL volume: 17 µL of Aqua dest. (nuclease free) are mixed with 2 µL appropriate 10x restriction enzyme buffer, 1 µg (1µg/µL) of DNA added and mixed well. Then 1 µL (10 U) of restriction enzyme (-20°C) are added and again mixed well by swirling and spinning down. After incubation time of 1 hour at 37°C in a water bath (or other appropriate temperature according to manufacturers protocol) DNA fragments can be resolved by agarose gel electrophoresis.

For a double enzyme digest consult the website of the manufacturer and check for appropriate conditions for simultaneous double digest or alternatively digest at the low salt buffer with enzyme 1 first (1 h), then add NaCl to reach the salt concentration of the second enzyme, using e.g. the mixing cross at: (Link), add enzyme 2 and continue with the digest for another hour.

 

Ligation

In a microcentrifuge tube prepare the following reaction mixture:

Linear vector DNA: 5-10 µl (50-400 ng)

Insert DNA: use a 1:1 up to a 3:1 (or 5 :1) molar ratio of insert DNA to vector DNA

10X ligation buffer for T4 DNA Ligase : 2 µl

50% PEG 4000 solution (for blunt ends only): 2 µl

Water, nuclease-free     to 20 µl

T4 DNA Ligase: 0.2-0.4 µl (1-2 u) for sticky ends or 1 µl (5 u) for blunt ends

Vortex the tube and spin down in a microcentrifuge for 3-5 seconds.

Incubate the mixture for 1 hour at 22°C.

After ligation: heat to 65°C for 15 min to destroy DNA-Ligase complexes

(increases transformation efficiency 3-5 fold)

Use the mixture for transformation.

Note

If the yield of ligation product is insufficient, prolong the reaction time (overnight).

DNA can be dissolved in nuclease-free water or TE buffer: 10 mM Tris-HCl, 1 mM EDTA (pH 7.8). An excess of ligation mixture with respect to competent cells may decrease the transformation efficiency.

Hints from Fermentas:

Insert : Vector = 5 : 1 (related to pmol ends)

for 10 ng vector (3 kb) with sticky ends:
use 1-2 units T4 DNA ligase (10 units for 100 ng)
for blunt ends: use 5 units for 10 ng (50 units for 100 ng)

  PEG4000 increases the ligase efficiency (but has to be removed for electroporation by 2x chloroform extraction)

ammonium inhibits the ligase (consider that for PCR products containing NH4+)

NaCl > 200 mM inhibits the ligase

 

Agarose Gel Electrophoresis

Solutions:

TAE 50x stock: Tris base 2 M, 57.1 mL acetic acid, EDTA 50 mM (stock 1 M pH 8), ad 1 L with AD

Ethidiumbromide 10 mg/mL in aqua dest.

Running buffer: TAE 1x

DNA sample buffer 6x:
(Fermentas:10mM Tris-HCl (pH 7.6), 0.03% bromophenol blue, 0.03% xylene cyanol FF, 60% glycerol, 60mM EDTA

Typically, 1 % agarose gels are poured by heating up 1 g agarose / 100 mL 1 x TAE until agarose is completely dissolved and no smears are visible. The hot agarose solution is cooled down to about 55°C and 5 µL ethidium bromide solution are added and gently mixed to avoid formation of bubbles. Then, the agarose solution is poured immediately into the gel cast cassette, appropriate combs are placed in the solution and abundant bubbles are pushed away from the slots by pipette tips. After about 30 minutes, polymerization is finished and the agarose gel can be used for electrophoresis. The agarose gel is placed in the electrophoresis chamber (BioRad) with the slots facing the cathode and 1x TAE is added until the gel is sufficiently covered by running buffer. If necessary, the slots are rinsed with running buffer by pipetting up and down some running buffer in order to remove leftovers of agarose slurry. DNA samples are mixed with 6x DNA sample buffer (5:1) and loaded in the slots. After loading, electrophoresis is started immediately in order to prevent diffusion of sample into the surrounding gel. Electrophoresis parameters (power supply BioRad Power PAC 2000): 100 – 120 V for at least 30 minutes depending on the DNA fragment size supposed being resolved. Resolved DNA fragments are visualized under UV transillumination (transilluminator: 260 – 340 nm range) and documented with a BioRad gel doc system. For clonings care is taken that the DNA is not exposed too long to UV light since UV light can irreversibly damage DNA. DNA fragments are excised with a clean razorblade and gel extraction is carried out as described below.

 

Gel Extraction

Solutions:

Solubilization buffer (QG)

Isopropanol

Wash buffer (PE)

Elution buffer (EB): Tris/HCl 10 mM pH 8.5

For gel extractions usually the QIAquick gel extraction kit is used (Qiagen, Cat.no. 28704). An excised agarose gel slice is weighed to calculate the approximate gel slice volume (1 g = 1 mL) and transferred into an reaction tube. 3 volumes of solubilization buffer are added and incubated in a 50°C waterbath until the gel piece is completely dissolved. 1 gel volume isopropanol is added and mixed. The solution is then loaded on the column and centrifuged for 1 minute at full speed (centrifuge: Eppendorf centrifuge 5415 D), the flow through is discarded. 750 µL of wash buffer are pipetted to the column and centrifuged under the same conditions as above, the flow through is discarded and the column is placed in a new microreaction tube. For elution of extracted DNA, 20 µL of elution buffer are directly applied on the matrix, incubated for 1 minute at room temperature and centrifugation as above is carried out. As an alternative to the Qiagen gel extraction kit, sometimes GenElute columns (Sigma, cat. no. # 5-6500) are used, which are equilibrated by loading of 100 µl 1x TE buffer, followed by brief centrifugation (1 min at full speed), addition of the gel slice to the column and centrifugation at full speed for 10 min. The resulting flow through (about 40 µl) contains about 90 % of the DNA, while the agarose remains in the column.  

 

PCR: Polymerase Chain Reaction

Preparative PCR (e.g. for Cloning)

Solutions:

  •  AD nuclease free

  •  dNTP mix, each nucleotide 2 mM: stock solutions 100 mM

  •  Forward and reverse primers 10 pmol/µL in AD respectively

  •  10x Pfu puffer (or Taq buffer)

  •  Template DNA

  •  Pfu polymerase 2.5 U/µL – or Taq polymerase 2.5 U/µL (Pfu is used for cloning purposes due to the higher fidelity of this DNA polymerase; Taq polymerase is used for analytical PCR’s)

 

For a 100 µL PCR reaction, following solutions are mixed in a thin walled reaction tube (BioRad): 85 µL AD, 10 µL Pfu buffer 10x, 1 µL dNTP mix 2 mM, 1 µL forward primer 10 pmol/µL, 1 µL reverse primer 10 pmol/µL, 500 ng (1 µL) template DNA, 1 µL Pfu polymerase (2.5 U). Handling and pipetting of PCR steps are carried out with gloves in order to prevent contaminations. The reaction tubes arere placed in the hot (94°) PCR block (Perkin Elmer Gene Amp PCR System 2400) and the following program is used:

step 1   10 minutes 94°C (denaturation)

step 2   1 minute 94°C (denaturation)

step 3   1 minute 56°C or other appropriate annealing temperature – about 2-4 °C below the calculated melting temperature (annealing)

step 4 1.5 minutes/ 1kb 72°C (elongation)

step 5   5 minutes 68°C

step 6   4°C (cooling)

30 cycles of steps 2-4 are carried out. After finished PCR, 6x DNA sample buffer is added to the PCR samples to inactivate the polymerase. Amplified PCR products are resolved by 1 % agarose gel electrophoresis.

 

PCR-cloning

1. PCR with Pfu-Polymerase (has higher accuracy than Taq)

for 100 µl:

A.dest. 85 µl

10x Pfu-Buffer 10 µl

dNTP´s (20 mM)       1 µl (ad 200 µM)

Template-DNA          1 µl (50 ng)

Primer forward 1 µl (10 pmol, about 100 ng)

Primer reverse 1 µl (10 pmol, about 100 ng)

Pfu-Polymerase          1 µl (2.5 - 3 units)

94°C 1 min

94°C  1 min

annealing temp. 1 min

75°C 1 min/kb Insert

68°C 5 min

4°C unlimited

Take 10 µl of the PCR-Mix, add DNA-Buffer (2 µl 6x conc.) and run an agarose gel

(100 V, 30 min) - the PCR-product should be clearly visible.

 

2. Phenol/chloroform extraction and ethanol precipitation

The aqueous phase of the PCR-Mix (under the paraffin layer) is transferred to a new tube

(90µl), an equal volume phenol (or phenol:chloroform:isoamylalcohol=25:24:1) is added (90

µl) and the sample is vortexed for about 10 sec, followed by centrifugation (14000 rpm,

Eppendorf Centrifuge, 3 min).

The aqueous phase (upper layer) is carefully taken up with a pipette (using the “screwing

mode” for the last few µl). The white material between the two phases should not be taken up.

An equal volume chloroform (or chloroform:isoamylalcohol=24:1) is added, followed by

vortexing for 10 sec and centrifugation at full speed for 10 sec. The upper phase is again taken

up carefully and transferred to a new tube.

The volume of the final DNA-solution should be measured with the pipette and the DNA

precipitated by mixing with 1/10 volume 3M Na-acetate and 2.5 volumes absolute ethanol.

The solution should be cooled to -70°C for 5 min and the DNA pelleted by centrifugation

(14000 rpm, 30 min at 4°C). The supernatant is removed, the pellet is washed with 100 µl

70% EtOH and centrifuged again for 4 min (14000 rpm, 4°C). The washing solution is

removed, the pellet is dried for 5 min at r.t. and resolved in 10 µl TE-buffer (by resuspending

with a pipette and warming to 58°C for 5 min).

If possible, the amount of DNA is quantified by photometry (1 µl in 300 µl A.dest.).

A 260  x 15 = µg/µl (for dilution of 1:300 and dsDNA)

 

3. Restriction digest and electrophoresis

1 - 2 µg of the vector DNA is digested with the restriction enzymes of choice using the

appropriate buffer system). Usually a 20 µl solution is prepared (2 µl of 10x buffer, 1 - 2 µl of

DNA and A. dest. up to 20 µl). If two enzymes are used, which require different buffers (e.g.

L and H buffer), you can start with the low salt buffer and just 1 enzyme (1 µl enzyme, 10

units, 37°C for 1 h), followed by addition of NaCl to obtain the higher salt concentration,

addition of the second enzyme and incubation for an additional hour at 37°C.

If you use two different restriction enzymes for cloning (generating incompatible DNA-ends),

it is not necessary to dephosphorylate the vector. If you use just one enzyme (or enzymes with

compatible ends), you have to dephosphorylate the vector after the restriction digest (by

adding 1/10 volume of 10x CIP-buffer and 1 µl of CIP (calf intestine phosphatase) and

incubation at 37°C for 15 min. This is necessary to prevent religation of the vector (and a high

background in empty vectors after transformation).  The phosphatase has to be inactivated

afterwards by addition of EDTA (1 µl 0.5 M) and heating to 68°C (for 10 min). Phosphatase

treatment usually decreases the efficiency of ligation considerably.

The PCR-product should be subjected to restriction digests as the vector (use an appropriate

amount of DNA - e.g. 1 - 2 µg, as well). For the final ligation, you have to consider the molar

amount of insert related to vector. Since, the insert is usually much smaller than the vector

(e.g. 1 kb compared to 5 kb), 1 µg of insert means a corresponding higher molar amount (5x

more than 1 µg of a 5 kb vector).

After restriction digest (and dephosphorylation of the vector) the samples are either extracted

with phenol/chloroform and precipitated with ethanol as described above, or subjected to

electrophoresis. (Use low melting agarose, if you want to purify the DNA from the cut gel

slices by heating and phenol/chloroform extraction, or normal agarose if you want to use

GenElute columns from Sigma or similar stuff to obtain the DNA from the gel.).

Electrophoresis is done at 100 V for about 30 min (for normal agarose) or at 80V for 1 h (for

low melting agarose).

After electrophoresis, DNA-bands of linearized vectors or PCR-fragments are cut from the gel

(as close to the DNA as possible) preventing longer UV-exposure (which damages the DNA).

Gel slices can be transferred to GenElute columns (equilibrated with 100 µl TE and

centrifuged at full speed for 5 sec), followed by centrifugation at 14000 rpm for 10 min at r.t.,

which results in the generation of about 40 µl of DNA solution with about 80 - 90% of the

original DNA amount. Alternatively, the gel slices are transferred to Eppendorf tubes,

centrifuged briefly (to estimate the volume of the gel), covered with an equal amount of TE,

heated to 65°C for 5 min, cooled for 30 sec (at r.t.) and extracted with an equal volume of

phenol/chloroform as described above.

 

4. Ligation

1 µl 10x ligase-buffer, linearized (dephosphorylated) vector (about 100 - 300 ng), insert

(about 50 - 100 ng, depending on length) and A. dest (ad 10 µl) are mixed and ligase is added

(e.g. from BioLabs, 0.5 - 1 µl). The molarity of the insert should be between 1 - 2 times the

molarity of the vector . The ligation mix is incubated at 16°C over night (or alternatively: 4 h

at r.t.). The ligase buffer contains ATP (prevent repeated freeze-thawing; you can aliquot the

buffer and store it at -70°C for good results).

 

5. Transformation:

Transform 2 µl of the ligation mix (either conventionally or by electro-

transformation: 1.9 kV, 25 µF, 200 , 4-5 msec), recover for 1 h at 37°C in 1 ml SOC medium,

centrifuge at 3000 rpm for 10 min, decant the supernatant, suspend the pellet in the residual

fluid and streak out on appropriate plates.

Bacterial colony PCR

(to test for successful cloning and presence of inserts)

Ø  Mix a single colony in 20 µl A. dest

Ø  Heat to 95°C for 10 min (to lyse cells and release plasmids)

Ø  Spin at 14000 g for 2 min

Ø  Take 2 µl per PCR

PCR Mastermix (20x)

 

Per sample

For 20 samples

10x buffer (incl. Mg)

2 µl

40

2 mM dNTPs

2 µl

40

Primer 1

0.4 µl (40 ng or 4 pmol)

8

Primer 2

0.4 µl

8

A.dest

12.7 µl

254

Taq (Roche)

0.5 µl (0.5 u)

10

Supernat from colony

2 µl

 

 

PCR: A) 3 min 94°; B) 35 cycles: 30 sec 94°, 30 sec 52°, 45 sec 72°; C) 68° 5 min

 

Northern Blotting

1. Extraction of RNA

1.1.       Incubation of cells under the appropriate conditions (usually on petri-dishes with 10 cm diameter)

1.2.      Complete removal of the media with a sterile pasteur pipette linked to the vacuum pump: after the first removal of the media lean the petri-dishes nearly vertically against a support (for a short time) so that the rest of the media can be collected from the bottom (in the laminar flow).

1.3.      Add 1 ml of TriZol Reagent (Gibco: 15596-018) to the petri-dish and cover quickly the whole area (by shaking) - use sterile tip (if possible with a filter included).
This step should be done in the fume cupboard because of the phenol of the reagent. If you want to prevent any potential contamination with RNases, you can do it in the laminar flow, too.

1.4.      Extract the cells by repeated pipetting of the solution over the whole area (the solution usually gets a little bit turbid) and transfer the extract to a sterile eppendorf tube.

2.    Preparation of total RNA
according to the protocol provided by Gibco.

3.    Quantification of the RNA
The final pellet is dried for 10 min in the laminar flow and dissolved in 22 µl of nuclease-free distilled water by heating to 56°C for 30 min. 2 µl of the solution are diluted with 500 µl of distilled water and the A260 and A280 values are determined using quartz cuvettes (switch on the reader at least 1 h before in order to warm up the lamp).
40 µg RNA/ml have an A260 = 1
µg RNA/ml = measured A260 x 250 (dilution factor) x 40; thus: A260 x 10 = µg RNA/µl
The ratio A260/A280  should be 1.8 - 2.0 for clean RNA solutions.

4.    Dot Blot (otional):
Calculate the amount of RNA solution to give 5 µg of RNA (for higher sensitivity: 10 µg).
The volume has to be 3µl or less. Pipette the corresponding volume into a fresh sterile eppendorf tube and add nuclease-free water to give a total of 3 µl. Centrifuge the tubes to get a drop at the bottom of the tube. Then directly pipette 3 µl onto a dry Hybond-N+ membrane (you can draw a grid with 1 cm squares onto the membrane using a pencil and apply the drops in the middle of each square). Let the membrane dry for 10 min and then wet it a little bit from below using 5x SSC buffer. Immobilise the RNA on the membrane by UV-crosslinking (Stratagene Crosslinker - Dorian Bevec lab: Auto-crosslink with setting of 1200). Store the blot in Saran wrap at -20°C.

5.    Electrophoresis in denaturing Agarose-Gels
5x MOPS:    41.2 g 3-(N-morpholino-)-propanesulfonic acid
                               800 ml 50 mM Na-Acetate solution (4.1 g/l in nuclease-free water)
                               10 ml 0.5 M EDTA solution
                               adjust the pH to 7.0 with 2 N NaOH
                               adjust the volume to 1 l with nuclease-free water
                               autoclave the buffer and store at R.T. protected from light (wrapped in foil)

Preparation of the gel (1% Agarose)
1.5 g Agarose (Pharmacia, NA-Agarose) are suspended in 93 g of nuclease-free water (fresh bottle!) and heated for 3 min at full power (850 W) in the microwave oven. Shake a little bit to dissolve completely; weigh the amount of water that is lost due to evaporation and fill up to the original weight (if necessary heat again to dissolve completely and check the weight again).
Add 30 ml 5x MOPS buffer, 27 ml 37% formaldehyde (2.2 M) and 7.5 µl ethidiumbromide solution (10 mg/ml) - mix and try to prevent air bubbles.
The gel is poured into the gel-bed (this should be completely horizontal - check with bubble of spirit level) and the sample comb is put in (the sample comb is usually stored in ethanol to prevent RNase contamination and dried in the laminar flow before use). Polymerisation of the gel is allowed for 30 min and then the gel is transferred to the electrophoresis apparatus (filled with 1 l of 1xMOPS - diluted from 5x MOPS with nuclease-free water). The surface of the gel should be covered. The sample comb is carefully removed.
Preparation of RNA-samples for electrophoresis
10 µg total RNA (calculated volume)
2.5 µl 5x MOPS
3.5 µl 37% formaldehyde
10 µl formamide
are combined in an eppendorf tube, briefly centrifuged and the RNA is denatured by heating to 56°C for 15 min. After a brief cooling on ice, 2 µl of 10x loading buffer (50% glycerol, 1 mM EDTA, 0.4% bromophenolblue, 0.4% xylenecyanol) are added and the samples are again briefly centrifuged.
Electrophoresis
The samples are mixed with loading buffer (at the bottom of the tube after centrifugation) by repeated pipetting. Then they are carefully filled into the sample pockets of the gel. Electrophoresis is carried out at 20V over night or at 100V for 3 h (RNA migrates towards the plus-pole). The bromophenolblue should migrate about 8 cm, before ending the run. The gel is checked on the UV-monitor and a picture is taken with the Polaroid camera (put a ruler to the gel). Two bands should be visible (28S and 18S rRNA). Partly RNase-digested samples migrate faster.

6.    Capillary Blot
The gel is marked on the right lower edge, removed from the electrophoresis container and submersed in 0.05 N NaOH for 20 min (with some shaking). This is important for the transfer of RNA larger than 2.5 kb.
Wash the gel with nuclease-free water.
Shake the gel for 45 min in 20x SSC-buffer.
A tray is filled with 5x SSC, and a glass plate is put on the tray. Two Whatman 3MM filters (11 cm x ca. 40 cm) are wetted with the 5x SSC and laid over the pate so that both ends are in the buffer. Air bubbles between the plate and the filters are removed by rolling a sterile pipette on the filter. The gel is put on the wet filters with the upper side down (transfer is more efficient in this way; besides, the orientation of the samples on the blot is then equal to the orientation on the gel). Air bubbles between gel and filters have to be removed with the pipette !!!! A pre-cut Hybond-N+ membrane (10 cm x 14 cm, dry) is put exactly on the gel (the membranes becomes wet when it touches the gel).
2 Whatman 3MM filters (10 cm x 14 cm) are wetted in the 5x SSC buffer and laid on the Hybond-membrane. Air bubbles are removed again. (If the pre-cut 10 cm x 13 cm Whatman filters are used, the gel and the Hybond membrane have to be cut to this size; in this case just 13 instead of 15 samples can be applied to the gel).
Parafilm is put exactly to the edges of the gel, in order to prevent contact between the wet filters below the gel with the filters above the gel.
A stack of dry Whatman filters (height: 5 - 8 cm) is laid on top, followed by a glass plate and a 500 g weight (bottle). The capillary transfer from the gel to the membrane should be carried out for 18 - 24 h. Afterwards, the transfer is checked under UV-light. There should be no bands visible on the gel, but only on the Hybond-membrane (a picture can be taken with the Polaroid camera)

 

Fig. 1: Capillary Blot
The RNA is immobilised on the membrane by UV-crosslinking (Exposure to 120 mJ; Bevec´s lab: Stratagene Auto-Crosslink set to 1200 units).
 

7.   Methyleneblue staining of the Blot (optional)
Wash the membrane for 10 min in 3% HAc (under nuclease-free conditions)
Stain for 30sec - 1 min with 0.04% methyleneblue/0.5 M Na-acetate pH5.2
Destain with nuclease-free dist. water until the background is nearly white.

8.   Pre-hybridisation
Reagents:   
50x Denhardt´s:       10 g Ficoll (Sigma F-9378)
                               10 g Polyvinylpyrrolidone (Sigma PVP-10)
                               10 g BSA (Sigma A-7906)
                               ad 1000 ml with nuclease-free water
Pre-hybridisation solution:   
5x SSC                               (25 ml 20x SSC)
5x Denhardt´s                   (10 ml 50x Denhardt´s)
20 mM Na-phosphate       (20 ml 1 M Na-phosphate buffer pH7.0)
7% SDS                              (35 ml 20% SDS)
                                          (10 ml nuclease-free water)
                                          (stored in aliquots at -20°C)

The pre-hybridisation solution is pre-warmed to 65°C before use.
Hybridisation solution
= pre-hybridisation solution containing 10% dextransulfate (10 g/100 ml)
(stored in aliquots at -20°C).
For the pre-hybridisation, the blots (wet) are put into the hybridisation tubes (not more than 2 blots per tube) with the RNA facing inside, and 10 ml of pre-warmed pre-hybridisation solution are added, as well as:
       100 µl Poly-A(10 mg/ml; final concentration 100 µg/ml)
       100 µl sonicated fish sperm DNA (10 mg/ml; final concentration: 100 µg/ml)
       (for 10 ml = for one tube)
The blots are pre-hybridised for 4 h at 65°C (2.5 h are sufficient, too) under continuous rolling of the tube.
1 h before the end of the pre-hybridisation, the hybridisation solution is pre-warmed to 65°C and the radioactive labelling of the oligo is carried out.

9.   32P-labelling of the oligonucleotide (with Terminal deoxynucleotidyl-Transferase)
1 µl Oligonucleotide solution (10 ng/µl) = 10 ng
2 µl 5x TdT reaction buffer
1 µl CoCl2 solution (10 mM)
1 µl TdT enzyme (included in the TdT-Kit: Boehringer Nr. 220582)
5 µl (-32P) dATP (50 µCi) (Amersham, Redivue)
Mix by centrifugation and incubate for 1 h at 37°C
In the meantime prepare NICK spin column (Pharmacia) for the purification of the labelled oligo: put the column in vertical position, let the gel settle and remove the lids. Let the column drain and apply 1 ml TE-buffer, let it drain again and apply 2 ml TE-buffer. Let the column drain again, put into a centrifugation tube and centrifuge it at 500 g for 4 min using a counterbalance (2000 rpm in the Heraeus centrifuge). Remove the water that was spun off, and put a small cryotube (1.5 ml) into the centrifugation tube, followed by the column. (see Fig. 2).
After the incubation of the oligonucleotide with (-32P) dATP and TdT, add 100 µl of TE-buffer containing bromophenolblue and mix shortly. Briefly centrifuge the sample and apply it to the prepared NICK spin column (the solution is taken up by the semi-dry gel). Centrifuge the column under exactly the same conditions as before (500 g, 4 min). Low molecular compounds like bromophenolblue or unbound (-32P) dATP remain in the gel, whereas high molecular compounds as the (-32P) dATP-labelled oligonucleotide are spun into the cryotube.

Fig. 2
 

10.  Hybridisation
The pre-hybridisation solution is poured off and 10 ml pre-warmed hybridisation solution is added, as well as 100 µl Poly-A solution and
100 µl sonicated fish sperm DNA
Finally the labelled oligonucleotide is added, and the tube is transferred to the hybridisation oven. Hybridisation is carried out over night at 65°C with continuous rolling of the tube. 

11.  Post-hybridisation washing
After the hybridisation, the radioactive solution is poured onto some paper towels on a bench coat and can be thrown to the solid radioactive waste. The membranes are removed from the tubes into a plastic container and washed with continuous shaking under appropriate conditions in a water bath. Two or three washing solutions are used, usually at 65°C for 20 min, each.
Oligo-Wash 1:         
150 ml 20x SSC
100 ml Na-phosphate buffer pH7.0
100 ml 50x Denhardt´s
400 ml nuclease-free water
250 ml 20% SDS (at last)

Oligo-Wash 2:
50 ml 20x SSC
900 ml nuclease-free water
50 ml 20% SDS (at last)                   = 1x SSC/1% SDS  (Na+-concentration: 0.225 M)

Oligo-Wash 3 (optional):
10 ml 20X SSC
980 ml nuclease-free water
10 ml 20% SDS                               = 0.2x SSC/0.2% SDS (Na+-concentration: 0.045 M)
The washing solutions are pre-warmed to the appropriate temperature (usually 65°C)
For each oligonucleotide the melting temperature has to be calculated for the different washing solutions according to the formula:
Tm = 81.5 + 16.6 log (Na+-concentration) + 0.41 (%GC) - 600/N - 0.63 (formamide%)
%GC.... percentage of G and C in the oligo
N ......... length of the oligo (number of bases)
(in our procedure there is no formamide included)

The last washing step should be about 10°C below the calculated melting temperature.
For oligonucleotides in the range of 30 bases, oligo-wash 1 and 2 are usually sufficient, for oligos in the range of 60 bases a third wash is recommendable. Either the temperature or the sodium concentration has to be adjusted in a way that the applied temperature is 10°C below the melting point.  After the use, the first washing solution has to be poured into the container for liquid radioactive waste, the second and the third washing solution can be poured into the decontamination sink. The membranes are wrapped in Saran wrap and either measured on the InstantImager detector or exposed to film (Kodak X-OMAT or BioMax). The film cassette is stored at -70°C until development of the film.

12. Dehybridisation:
After evaluation of the northern blot, the membrane can be stripped in reprobed. The stripping is carried out by washing in 0.1% SDS at 80°C for 20 - 30 min. The next probing starts with the pre-hybridisation.