E Coli Transformation Protocol

(This is a general protocol that works with most competent cells. If using commercially prepared competent cells, it‘s always good to check the product insert and protocol.)

1. Thaw an aliquot of cells on ice.

2. Mix ~ 1 μL DNA (in H2O or elution buffer) and 25 μL cells.

3. Leave on ice for 10-15 min.

4. Heat shock at 42 °C for 45 seconds.

5. Place back on ice for 2 minutes.

6. Add 250 μL of SOC and incubate at 37 °C for 1 hr.

7. Plate 100 μL on LB plate (with appropriate antibiotic) and grow overnight at 37 °C. 

S.O.C Medium (adapted from Invitrogen)

For 100 mL:

2 g tryptone (2% tryptone)

0.5 g yeast extract (0.5% yeast extract)

10 mM sodium chloride (200 μL 5 M NaCl) 2.5 mM potassium chloride (250 μL 1 M KCl) 10 mM magnesium chloride (1 mL 1 M MgCl2) 10 mM magnesium sulfate (1 mL 1 M MgSO4) 20 mM glucose (1.8 mL 20% glucose) 

Method For Making Competent C43De3

Preparation of CaCl2 competent cells of C43(DE3)
„h Streak the strain from a LB agarose stab or a seed stock vial stored at -80¢XC onto LB plate.
„h Inoculate a single, well-isolated colony into 5 ml LB medium in a 50 ml tube.
„h Incubate 24 hours at 37¢XC, shaking at 250 rpm.
„h Inoculate 100 ml of LB medium, in a 500 ml Erlenmeyer flask, with 1 ml of the saturated overnight culture.
„h Grow this flask at 37¢XC, shaking at 250 rpm, until the OD600 reaches 0.6-0.8.
„h Transfer the culture to two 50 ml tubes, and place the tubes on ice for 10 minutes.
„h Centrifuge the tubes at 4,000 rpm for 10 minutes at 4¢XC.
„h Discard ALL the supernatant, and resuspend the pellet GENTLY in 10 ml of ICE-COLD 0.1 M CaCl2 / 10% glycerol. Put the tubes back on ice for 15 minutes.
„h Centrifuge the tubes again at 4,000 rpm for 10 minutes at 4¢XC.
„h Again, discard ALL the supernatant, and resuspend the pellet GENTLY in 1 ml of ICE-COLD 0.1 M CaCl2 / 10% glycerol.
„h Prepare aliquots 50 £gl in individual Eppendorf tubes, on ice. Freeze immediately the aliquots and store them in a -80¢XC freezer.

Preparation Of Chemically Competent C43 De3

Can be used for preparation of other strains of chemically competent E. coli
(based on Qiagen protocol)
Day 1.
1. Start with a glycerol stock of C43 (DE3) strain. Streak out E. coli on LB plate and grow overnight at 37 °C. C43(DE3) strain has no antibiotic resistance, so no antibiotic should be included at this point. Once cells are made competent and are used in transformation, the choice of antibiotic will depend on the selective marker present on the plasmid that is being introduced into the C43(DE3) cells.
2. Prepare the following buffers and store them at 4 °C until they are needed.
Also, prepare and sterilize LB medium, centrifuge tubes, pipette tips and microcentrifuge tubes.
100 ml TfbI buffer:
30 mM CH3COOK 3 ml 1 M stock (9.8 g/100ml)
50 mM MnCl2 5 ml 1 M stock ( 19.8 g/100 ml)
100 mM KCl 10 ml 1 M stock (7.5 g/100 ml)
KCl can be substituted with RbCl
10 mM CaCl2 1 ml 1M stock
150 g/L glycerol 15 g
Carefully pH to 5.8 with 0.1 M acetic acid (0.575 ml of glacial CH3COOH/100 ml of H2O). Discard if you overshoot the pH, or a precipitate will form which does not redissolve and the buffer does not work.
 
Mix all components together, adjust pH, and filter sterilize the buffer using a 0.2 μm syringe filter.
10 ml TfbII buffer:
10 mM MOPS-Na 1 mL 100 mM stock (2.09 g/100 mL, pH 7.0 with NaOH)
75 mM CaCl2 0.75 mL 1 M stock
10 mM KCl 0.1 mL 1 M stock
150 g/L glycerol 1.5 g
Mix all components together and filter sterilize the buffers using a 0.2 μm syringe filter.
 
Day 2.
1. Grow a single colony from the plate for 2 hr in 5 mL of LB (or TY broth, 2x YT, etc – any rich medium will do), at 37 °C, shaking at 250 rpm. Don‘t include any antibiotics. Strain C43(DE3) has no antibiotic resistance genes.
2. Transfer to a flask with 100 mL of LB medium and grow until OD550= 0.5-0.6 (~2-3 hrs)
3. Spin cells in sterile centrifuge tubes at 2000 x g for 5 min, at 4 °C.
a) The most convenient way to do this is to use the refrigerated benchtop centrifuge, Sorvall Legend RT, and spin cells in 50 mL conical tubes (they are already in a sterile package).
b) Another option is to use JA 25.50 rotor in Beckman Coulter Avanti J-25 centrifuge and spin cells in Beckman 50 mL centrifuge tubes (polycarbonate or polyallomer). These tubes must be sterilized prior to use either by autoclaving or some other suitable method. Depending on the material, some tubes may not be able to withstand autoclaving (please consult Beckman product information sheets provided with tubes for sterilization instructions).
4. Carefully pour off the supernatant and gently resuspend cells by slowly pipetting up an down the side of the tube. Resuspend cells in total of 40 mL of cold TfbI. NEVER VORTEX when making competent cells or working with competent cells. Keep cells on ice while resuspending.
6. Leave on ice for 10 min.
7. Spin cells as gently as possible, at 2000 x g, at 4 °C.
8. Remove supernatant and gently resuspend the pellet in total of 4 mL of sterile TfbII. Keep cells on ice while resuspending.
9. Aliquot 25-50 μL cells into sterile microcentrifuge tubes. Freeze in liquid nitrogen and store at -80 °C.
The cells are now chemically competent and ready for transformation

Protocol For Northern Analysis

Gel Electrophoresis
Prepare formaldehyde-agarose gel. Add 65 mls of DEPC-treated water to 1.08 g of Seakem LE
agarose and microwave until agarose dissolves completely. Allow solution to cool to 60ºC, then
add 9 mls of 10X MOPS and 16 mls of 37% formaldehyde. Swirl to mix and pour into gel box (9
x 11 cm).
• Transfer 0.5 – 2 mg of total RNA to a new tube and dry samples by speed vacuum (it is
not necessary to dry).
• Resuspend dried samples in 18 ml of loading buffer. Add 2 ml of 10X tracking dye, 0.05
ml of EtBr, and 1 ml of glycerol.
• Heat RNA samples to 65ºC for 10 minutes, cool on ice for ~1 minute and then do a quick
spin down of the samples.
Prepare 1XMOPS running buffer (50 mls of 10X MOPS and 450 mls of DW water).
Place gel in gel box and submerge in running buffer.
• Load RNA samples onto gel and run at 30-50 V until bromophenol blue (fastest
migrating of the two tracking dye bands) migrates ~ 60-75% of the total distance of the
gel.
 
Transfer RNA from gel to nylon membrane
• Rinse gel in DW 6 times, 5 minutes each. Then rinse once for 5 minutes in 20X SSPE.
• Cut membrane so that it is the exact size of the gel. Rinse membrane once in DW then
soak in 20X SSPE for 5 minutes.
• Assemble transfer apparatus per Maniatis Laboratory Manual:
1. Cut a wick that is the exact size if the gel. Cover the platform of the gel box with
the wick so that there is an even amount of the wick in each of the wells. Fill the
wells with 10X SSPE.
2. Add parafilm to the sides of the gel box so that the solution does not evaporate
during the transfer.
3. Flip gel over and remove any air bubbles that are trapped underneath of it.
4. Place the membrane on top of the gel. Try to position correctly so that you do not
have to adjust the membrane at all as RNA transfers immediately. Again remove
any air bubbles.
5. Place 3 pieces of Whatman paper that are larger than the membrane and that have
been pre-soaked in 2X SSPE on top of the membrane.
6. Then sack ~25-50 paper towels on top if the Whatman paper. Lastly place a heavy
object on top of the paper towels. For diagrams see the Maniatis Manual.
7. Transfer for 16-20 hrs.
 
After the transfer, mark the blot with a pencil. To do this, carefully remove the paper
towels and the Whatman paper so as not to disturb the gel or the membrane. Flip the
membrane and gel over and trace the wells with a pencil.
• Wash membrane in 20X SSPE for 30 minutes, then soak in 2X SSPE for 5 minutes. Wrap
the membrane in plastic wrap and cross-link by UV.
• Stain blot with methylene blue by successively washing the blot in 2X SSPE for 5
minutes; 1N acetic acid for 5 minutes; 0.04% methylene blue in 0.5 M sodium acetate
(pH 5.2) for 5 minutes; rinse in DW until most of the membrane is destained except for
the ribosomal bands. The blot may be wrapped and stored @ -80ºC at this point,
desiccated at 4ºC for months or probed immediately.
 
Northern Probe Preparation
• Begin to prepare probe template by making desired DNA fragment by PCR and/or
restriction digest of larger fragment. Isolate fragment by gel purification and gene clean
treatment (per manufacture suggestions). Fragments can be many different sizes although
fragments that contain several hundred base pairs often work best.
• Use 25-50 ng of DNA in 1X TE. Boil for 10 minutes, quickly chill on ice and then spin
down.
• I have been using Amhersham’s Ready-to-go DNA labeling beads (-dCTP). Their
protocol is very easy to follow.
• After the 5-15 minute incubation remove unincorporated labeled nucleotides using
Amersham’s ProbeQuant G-50 micro columns (again a very easy protocol that is
supplied with the columns).
• Test % incorporation by spotting 1 ml of the probe onto a DE-81 Whatman filter circles.
Repeat this and wash this one with 0.5M phosphate buffer and count.
 
Hybridization
• Prehybridize the blot in 5-10 mls of Stratagene’s Quickhyb solution per 11 X 14 cm
membrane at 42ºC in Northern oven for 30 minutes or longer.
• Add 50 ml of salmon sperm and then boil for 10 minutes, chill on ice, and add the whole
probe to the prehybridized blot. Incubate at 65-70ºC for 16-24 hrs.
• Remove hybridization solution and wash blot:
1. Twice in 6X SSPE/0.5% SDS for 15 minutes @ RT.
2. Twice in 1X SSPE/0.5% SDS for 10 minutes @ 37ºC.
3. Once in 0.1X SSPE/0.1% SDS for 30 minutes @ 65ºC.
• Wrap blot in plastic wrap and expose to film and/or phosphoimager.
 
Stock Solutions
10X MOPS
0.2M MOPS (42 g)
50 mM anhydrous sodium acetate (4.1 g)
10 mM EDTA (20 mls of 0.5 M)
pH to 6.5 and bring to a total volume of 1L with DEPC-treated water
RNA Sample Loading Buffer for 5 samples for 15 Samples
50 ml formamide 150 ml formamide
18 ml formaldehyde (37%) 54 ml formaldehyde (37%)
12 ml DEPC water 36 ml DEPC water
10 ml of 10X MOPS 30 ml of 10X MOPS
10X Tracking Dye
0.1% Bromophenol blue
0.1% Xylene cyanol
Dissolve in DEPC water
20X SSPE
175.3 g of NaCl
27.6 g of NaH2PO4*H2O
7.4 g of EDTA
Adjust pH to 7.4 and bring volume up to 1L 

Preparation Of Poly A Rna And Northern Analysis

Procedure  Use RNase-free tips and reagents and wear gloves:

 

1) Remove organs from six C57Bl mice (do cervical dislocation immediately before organ removal) and freeze directly into liquid nitrogen. Store at -70°C.  For lacrimal gland, in which mRNA yields directly from the organ have often been low, a better approach is to do an isolation of lacrimal acinar cells from six C57Bl mice (see method in Lab Protocols; leave out Percoll step) first, then extract mRNA.  To extract from cultured cells (ie. HT1080), begin with one T75 flask (approx. 2 x 106 cells).

 

2)  Have Pharmacia kit reagents warmed to RT for 30 min.  Check if Extraction buffer is in solution; if not warm to 37°C then cool to RT.  Have water bath at 65°C and 0.5 ml/purification of elution buffer warming at 65°C.  Resuspend Oligo(dT) and pipet 1 ml aliquots into individual microcentrifugation tubes (1 ml/purification).   Weigh out 0.1 – 0.23 gm of tissue, and place in a 7 ml mortar (alternatively, can use a polytron).  Add  0.4 ml of extraction buffer; use pestle to homogenize.  Add 0.8 ml of elution buffer, mix, homogenize and transfer to a clean 1.5 ml  microcentrifugation tube.  Centrifuge for 1 min (RT) full speed.  Centrifuge Oligo(dT) for 2 sec (just before gets to full speed, release button; spinning longer makes resuspension of Oligo(dT) difficult).  Discard supernatant from Oligo(dT) and add to it 1 ml of supernatant from tissue homogenization.  Mix for 3 min by inverting on rotor.

 

3) Spin 2 sec, discard supernatant.  Wash five times each with 1 ml of High Salt Buffer (quick spins between several inversions to mix).  Wash two times each with 1 ml of Low Salt Buffer.  Resuspend in 0.3 ml of Low Salt Buffer and transfer to a Microspin Column (break off end before use) in a microcentrifuge tube.  Spin at full speed for 5 sec.  Discard effluent and add 0.5 ml of Low Salt Buffer.  Spin and wash two more times with 0.5 ml each of Low Salt Buffer.  To elute, place column in a fresh microcentrifuge tube and add 0.2 ml of prewarmed Elution buffer.  Spin at full speed for 5 sec.  Add a second 0.2 ml of prewarmed Elution buffer and spin again.  Place eluted mRNA on ice.

 

4)  To determine OD260 of mRNA, have a quartz cuvette soaking in conc. HCl/methanol (1:1) for 1 hr prior to use to remove RNase.  Rinse well with DEPC treated ddH2O.  Zero spectrophotometer with elution buffer then add all of mRNA sample to cuvette to determine OD260 and OD260/OD280.  For µg/ml RNA, multiply OD260 value times 40 (1 OD260 is 40 µg/ml).  For storage, precipitate mRNA with 10 µl of Glycogen solution, 40 µl of potassium acetate solution and 1 ml of -20°C 95% EtOH.  Store at -70°C.

 

5)  For Northern analysis, have heating block at 56°C and pour denaturing gel (mid-size gel: to 0.8 gm of agarose, add 78.4 ddH2O and 5 ml of 20 x E buffer; heat to dissolve; let cool to not too hot to touch, then quickly add 16.6 ml of 37% formaldehyde, swirl to thoroughly mix and pour).  Spin precipitated mRNA for 15 min (4°C, full speed).  Carefully remove and discard supernatant.  Wash pellet with RNase-free 80% EtOH, dry and make up in 20 µl of ‘Buffer A’.  Also have size standards including an RNA ladder (mix 20 µl of ladder + 28.5 µl of ‘Sample Buffer’) and 28S/18S rRNA standard (mix 20 µl of ladder + 28.5 µl of ‘Sample Buffer’).  Heat at 56°C for 15 min, then place on ice.  Add 4.9 µl of ‘Glycerol Stain’ to mRNA and 12.5 µl to standards.  Load onto gel immersed in 1 x E/formaldehyde buffer (see below) with standards in the outside lanes.  Run overnight at 35 V (constant).

 

         20  X E Buffer                              1 x E/Formaldehyde Buffer

Na2HPO4.7H2O       32.72 gm                      ddH2O                  773 ml

NaH2PO4.H2O         10.85 gm                      20 x E buffer       50 ml

 – ddH2O to 1000 ml –                               37% formald.       177 ml

 

         Sample Buffer                              Buffer A

formamide           145.2 µl                       DEPC ddH2O          118 µl

37% formald.       42 µl                            sample buffer      200 µl

20 x E buffer       12.8 µl

 

 

 

         Glycerol Stain                               DEPC ddH2O

DEPC ddH2O          7 ml                              Add 0.8 ml of DEPC

glycerol               3 ml                              (Sigma #D-5758) to 800 ml

bromophenol bl.    25 mg                           ddH2O.  Warm overnight at 37°

xylene phenol       25 mg                           then autoclave.

 

 

6) Carefully cut out the standards lanes and place in ethidium bromide (5 µg/ml; 5 µl of stock per 100 ml of ddH2O) for 30 min.  Wash overnight in ddH2O, then photograph on transilluminator with florescent ruler.  The rest of the gel (cut off the left bottom corner to aid in orientation) is transferred to a clean glass tray and immediately denatured in 900 ml of 50 mM NaOH/10 mM NaCl (to 893.7 ml of ddH2O add 4.5 ml of 10 N NaOH and 1.8 ml of 5 M NaCl) for 20 min on a rotator.  Carefully pour off and add 900 ml of 0.1 M Tris, pH 7.6 to neutralize (20 min on rotator).  Pour off and add 900 ml of 20 x SSC for 45 min with gentle rotation.  To keep gel immersed during above steps, can balance an inverted ddH2O filled 50 ml conical on it, making sure that the top surface of the gel is wet before placing on the conical. 

 

7)  During the above 20 and 45 min steps, preparations for the overnight transfer can be initiated.  Needed are: (i) a glass tray or lucite box filled to a depth of 2 cm with 10 x SSC, (ii) a lucite support for the gel (can invert the gel mold in the reservoir),  (iii) a long piece of 3 MM paper which stretches over the lucite gel support on either side into the 10 x SSC; wet with 10 x SSC, (iv) nitrocellulose (Amersham Hybond ECL) cut to the same size as the gel (wear gloves in this and all steps), (v) two pieces of 3MM paper cut to the same size as the gel; place one on the long 3 MM piece and wet with 10 x SSC, (vi)  a 6-8 inch stack of paper towels cut to the same size as the gel, (vii) a glass tray containing ddH2O, (viii) a glass tray containing 10 x SSC, (ix) two 10” x 12” pieces of X-ray film, and (x) flat forceps.

 

8)  To set up the transfer, sandwich the gel between the X-ray film pieces and carefully invert (do over the 20 x SSC).  Place back side up on the wetted gel-size 3MM paper (on inverted mold).  Remove any air bubbles by gently rolling a capless 50 ml conical over the gel.  Add some 10 X SSC to keep from drying out.  Completely surround the gel with Parafilm or plastic wrap.  Next, wet the nitrocellulose paper by floating on, then immersing in, the ddH2O.  Immerse the wet filter in 10 x SSC for 5 min.  Holding the filter by two sides, carefully set down exactly on the gel (can’t reposition).  Carefully roll the capless conical over the filter to ensure removal of any air bubbles.  Add the remaining gel-size piece of 3MM paper (wet with 10 x SSC) and roll with conical.  Carefully add the cut paper towels to a height of 6”. Balance a glass tray on top of the towels to weigh down; stabilize the tray with tape running down on all sides to the 10 x SSC reservoir.  Cover with plastic to reduce loss to evaporation.  If transfer is initiated in the morning, carefully change the paper towels (can reuse the top dry ones) before leaving at the end of the day.  Let the transfer go overnight.

 

9)  Have 80°C vacuum oven heating up.  Remove the paper towels and, with the gel/filter still in place, invert.  Use an indelible fine tip marker to mark on the filter the edges of the gel (including the cut corner) and the loading wells.  Record experimental number at the top.  Peel off the gel and stain for 45 min in 0.5 µg/ml ethidium bromide (then place in ddH2O and examine/photograph on transilluminator).  Place filter in 6 x SSC for 5 min to wash, then place on 3MM paper and dry for at least 30 min.  Place the dried filter between two pieces of 3MM paper and bake under vacuum for 1-2 hr.

 

10)  To carry out hybridization to filter, prehybridize filter for 4 hr at 42°C (in prehyb/hyb. plastic boxes; gently rotation) in a prewarmed solution of 30% formamide, 5 x SSPE, 0.1% SDS, 5 x Denhardt’s, 0.2 mg/ml sonicated denatured salmon sperm DNA.

 

         Prehybridization Sol’n                     Hybridization Sol’n

                  ddH2O         15.3 ml                                  ddH2O         15.3 ml

         Formamide           15 ml                   Formamide           15 ml

         20 x SSPE            12.5 ml                         20 x SSPE            12.5 ml

         20% SDS              0.25 ml                         20% SDS              0.25 ml

         50 x Denhardt’s   5 ml                              50 x Denhardt’s   5 ml

         Salmon Sp. DNA   2 ml                              Salmon Sp. DNA   2 ml

         Poly A                  5 µl                              Poly A                  5 µl

                                                                        32cDNA probe       50-100 µl

 

(note: Salmon Sperm DNA stock is 5 mg/ml; Poly A stock is 10 mg/ml; make up prehyb and hyb sol’ns in 50 ml conicals)

 

11)  During prehybridization, label cDNA probe (purified insert) by random priming method (Pharmacia #27-9251-01).  Reconstitute reaction mix by adding 20 µl of ddH2O and place on ice for 5-60 min. Make 1.8 µl of insert up in 25 µl of TE, pH 8 and denature for 3 min at 95°C.  Cool on ice for 2 min, then add diluted/denatured insert to reaction mix.  Behind lucite, add 5 µl of 32PdCTP (ICN #33004X). Carefully mix by pipetting up and down.  Incubate at 37°C for 15-30 min.  Purify probe using a Nick Spin Column (Pharmacia #17-0862-02) or equivalent (Qiagen #28304) and check cpm’s in Scintillation counter.  Denature probe (3 min at 95°C, on ice for 2 min) prior to addition to hybridization solution.  Hybridize overnight at 42°C with gently rotation.

 

12) Remove filter into 400 ml of room temperature 2 x SSC, 0.1% SDS (40 ml of 20 x SSC and 4 ml of 20% SDS made up to 800 ml in ddH2O).  Wash for 5 min on rotator.  Do two additional washes. Transfer filter to 400 ml of prewarmed (42 – 65°C; use 42°C for wheat-mouse and 65°C for mouse-mouse hybridizations) 0.1 x SSC, 0.1% SDS (4 ml of 20 x SSC and 4 ml of 20% SDS made up to 800 ml in ddH2O).  Wash for 20 min on rotator.  Do a total of three 20 min washes.  

 

13) Allow filter to dry on fresh bench paper, then place on Whatmann 3MM paper in a large x-ray cassette holder.  Cover with plastic wrap.  Under red light in the dark room, place x-ray film on, followed by an enhancing screen.  Close up and expose overnight at -70°C.

Northern Transfer Protocol

A. Formaldehyde agarose gel electrophoresis

Note: Formaldehyde vapours are toxic and casting this gel should be performed in a fume hood. The gel tank must be covered when in use.

1. Preparation of equipment and reagents

Soak the gel tanks, combs etc in 0.2 M NaOH for 15 minutes to destroy any contaminating RNases before rinsing in milliQ water. It is not necessary to use DEPC treated water.

2. Make up 10 x Northern running buffer (containing MOPS):

Final conc: For 500 ml:
0.2 M MOPS 20.93 g
10 mM EDTA 10 ml of 0.5 M
50 mM Na Acetate 8.33 ml of 3 M

The buffer needs to be brought to a pH of 7.0. For 500 ml of 10 X MOPS this means add approx 2 g of NaOH. Make up to 500 ml in DEPC-treated water [To DEPC-treat solutions, add 0.1% DEPC to the solution in a bottle which can be autoclaved. Mix the solution well and allow it to stand, with the cap tightly closed, for at least 30 minutes. Then loosen the cap and autoclave. This must be done in a fume hood as DEPC is very toxic. Note also DEPC is inactivated by water. Allow the DEPC stock solution bottle to warm to RT before opening to avoid condensation. Parafiln shut].

Autoclaving of the MOPS buffer is not necessary and turns the solution yellow. Store at 4¡C.

3. Cast the gel

Cast a 14 cm, 0.7-1.0% agarose gel, which requires at least 100ml of agarose solution. Note: ethidium bromide is not added to the gel but rather to the sample buffer (see below). A thin, low percentage agarose (0.6-0.7%) gel is critical for good transfer of large MW RNA's (>4-5kb).

For 125 ml:
1.25 g agarose (1% gel)
12.5 ml 10 x running buffer
102.5 ml DDW
6 ml 35 % formaldehyde (check that it is a fresh batch that doesn't contain precipitates)

 

Dissolve the agarose in the microwave, let the solution cool to less than 60¼C, then add the formaldheyde and cast the gel.

4. Prepare RNA samples for loading

RNA is stored at -80¼C as an ethanol precipitate. Determine the amount of RNA to be loaded in each well (e.g. 2ug of poly A(+) RNA). Based on your RNA yield estimations, precipitate the appropriate amount of RNA ethanol solution for at least 15 minutes at 13,000g at 4¼. Remove all supernatant and resuspend each sample in 12ul of sample buffer.

RNA sample buffer (prepared fresh from frozen stocks):

For 500ul:

50ul 10 x running buffer

250ul deionised formamide

90ul formaldehyde

108ul water (DEPC-treated)

2ul ethidium bromide (stock concentration, 10 mg/ml)

Heat the samples at 65¼C for 5 minutes then to each add 3ul of RNA loading buffer. This consists of 0.25% bromophenol blue, 0.25% xylene cyanol in 20% Ficoll in DEPC-treated water.

5. Running the gel

Fill tank with 1 X MOPS (prepare 1X with DDW straight from the milliQ). Early protocols added formaldehyde to the running buffer but this is unnecessary. Run the gel at between 100 to 200V for several hours, until the xylene cyanol dye front has migrated 3 to 4 cm into the gel and the bromophenol blue is about 2/3rds down the gel. Circulate the buffer from end to end every half an hour, especially if running the gel at 200V. When the RNA has run an appropriate distance, photograph the gel including a ruler aligned with the wells. Pre-soak the gel in 20 x SSC while setting up the transfer (about 15 minutes).

B. Northern transfer

1. Setting up the transfer

The physical set-up for Northern transfer is identical to that for Southern transfer (see above) except that 20 x SSC is used as the transfer buffer (Note RNA is hydrolysed in strongly alkaline solutions within seconds!) and the membrane we use is GeneScreen Plus. Prewet membrane in DDW the 20 x SSC.

2. Post-transfer handling of the membrane

After overnight transfer, the position and orientation of the wells is marked on the membrane (#1 etc.) and it is rinsed gently in 2 x SSC for 5 minutes before being air dried in the fume hood. The membrane is baked at 80¼C for 2 hours and is then ready for pre-hybridisation and hybridisation.

3. Hybridize in Aqua hyb. with 0.1% SDS, 100ug/ml herring testes DNA (see Reagents).

Hybridising Northern Blots With Riboprobes

This method is obtained from Bill Clouston's thesis and is essentially that described by Kalinyak and Perlman (JBC262:460-4, 1987) with the addition of preservative-free sodium heparin to reduce non-specific hybridization.

Method

  1. Run Northern in the usual way, transfer to Genescreen Plus in 20x SSC and bake in oven at 80ºC for 2 hours.
  2. Hybridization buffer is as follows:
50% Formamide
3x SSC
10x Denhardts'
10 mM phosphate buffer, pH 8.0
2 mM EDTA
0.1% SDS
200 ug/ml herring sperm DNA
800 U/ml preservative-free sodium heparin (DBL, 25,000U/5ml, Royal Melbourne Hospital Pharmacy)
  1. Pre-hyb membrane for 4-6 hours at 60ºC.
  2. Hybridize in fresh buffer for 18-24 hours at 65ºC – 20 ng of riboprobe to the bag in a small volume of buffer (eg. 3-5 ml). Riboprobes should prepared exactly as described for hybridization histochemistry. This can include hydrolysis, but this may or may not make a difference to the degree of background.
  3. Wash at high stringency, ie. 0.1X SSC, 0.1% SDS at 65ºC. Be careful to wash all formamide-containing hybridization buffer off at low stringency first, ie. use a 2x SSC wash initially then increase stringency progressively. I obtained a good signal with little background using this washing regimen but lost the signal when I washed at 75ºC.
  4. Expose against X-ray film using an intensifying screen at -70ºC for 24 hours or longer. I obtained a good signal after 36 hours, equal to that obtained with Northerns using cDNA probes after 96 hours exposure.

Northern Hybridization Of Rna Fractionated By Agaroseformaldehyde Gel

Overview

The protocol is divided into three sections:

  1. Electrophoresis of an RNA preparation under denaturing conditions in an agarose-formaldehyde gel
  2. Transfer of the RNA from the gel to a nylon or nitrocellulose membrane by upward capillary transfer
  3. Hybridization analysis of the RNA sequences of interest using a labeled DNA or RNA probe.

I. Agarose/Formaldehyde Gel Electrophoresis

Prepare gel: Dissolve 0.75 g agarose in 36 ml water and cool to 60 oC in a water bath. When the flask has cooled to 60 oC, place in a fume hood and add 5 ml of 10xE running buffer and 9 ml formaldehyde. Pour the gel and allow it to set. Remove the comb, place the gel in the gel tank, and add sufficient 1xE running buffer to cover to a depth of ~ 1mm.

Prepare sample: Adjust the volume of each RNA sample to 6 µl with water, then add 2.5 x 6 µl freshly prepared sample denaturation mix. Mix by vortexing, microcentrifuge briefly to collect liquid, and incubate 15 min at 55 oC. Cool on ice for 2 min, then add 2 µl loading dye mix.

Run gel: Run the gel in 1xE running buffer at 100 volt for 10 min, then at 65 volt for 90 min

II. Transfer of RNA from Gel to Membrane

Prepare gel for transfer: Place the gel in an RNase-free dish and rinse with changes sufficient deionized water to cover the gel for 4×20 min.

Transfer RNA from gel to membrane:

  1. Fill the glass dish with enough 20xSSPE.
  2. Cut 2 pieces of Whatman 1MM paper, place it on the glass plate and wet it with 20xSSPE.
  3. Place the gel on the filter paper and squeeze out air bubble by rolling a glass pipet.
  4. Cut four strips of plastic wrap and place over the edges of the gel.
  5. Cut a piece of nylon membrane (MSI, Catalog #N00HY320F5) just large enough to cover the gel and wetted in water. Place the wetted membrane on the surface of the gel. Try to avoid getting air bubbles under the membrane.
  6. Flood the surface of the membrane with 20xSSPE. Cut 5 sheets of whatman 3MM paper to the same size as membrane and place on top of the membrane.
  7. Put paper towels on top of the whatman 3MM paper to a height of ~6 cm, and add a weight to hold everything in place.
  8. Leave overnight.

Prepare membrane for hybridization: Remove paper towels and filter papers and recover the membrane and flattened gel. Mark in pencil the position of the wells on the membrane and ensure that the up-down and back-front orientation are recognizable. Rinse the membrane in 5xSSPE, then place it on a sheet of Whatman 3MM paper and allow to try. Place RNA-side-down on a UV transilluminator (254 nm wavelength), and irradiate for appropriate length of time.

III. Hybridization Analysis

Prepare DNA or RNA probe (>108dpm/µg):

The probe labeled with Ridiprimer DNA labelling system (Amersham LIFE SCIENCE):

  1. Dilute the DNA to be labelled to a concentration of 2.5-25 ng in 45 µl of sterile water.
  2. Denature the DNA sample by heating to 95-100 oC for 5 min in a boiling water bath.
  3. Centrifuge briefly to bring the contents to the bottom of the tube, and put on ice for 10 min.
  4. Add the denatured DNA to the labelling mix and reconstitute the mix by gently flicking the tube until the blue colour is evenly distributed.
  5. Add 5 µl of Redivue [32P] dCTP and mix by gently pipetting up and down. Centrifuge briefly to bring the contents to the bottom of the tube, then incubate at 37 oC for 30 min.
  6. The probe is purified using ProbeQuantTMG-50 micro columns (Amersham pharmacia biotech):
  7. G-50 micro column preparation. Resuspend the resin in the column by vortexing, loosen the cap one-fourth turn and snap off the bottom closure. Place the column in a 1.5 ml screw-cap microcentrifuge tube for support, then pre-spin the column for 1 min at 3000 rpm in an Eppendorf model 5415C.
  8. Place the column in a new 1.5 ml tube and slowly apply 50 µl of the sample to the top-center of the resin, being carful not to disturb the resin bed. Spin the column at 3000 for 2 min. The purified sample is collected in the bottom of the support tube.

Hybridization:

  1. Pre-hybridization: Wet the membrane in the 5xSSPE and place it RNA-side-up in a hybridization tube and add 5 ml pre-hybridization solution, then place the tube in the hybridization oven and incubate with rotation 6 hr at 42 oC for DNA probe or 60 oC for RNA probe.
  2. Hybridization: Double-stranded probe was denatured by heating in a water bath for 10 min at 100 oC, then transfer to ice. Pipet the desired volume of probe into the hybridization tube and continue to incubate with rotation overnight at 42 oC for DNA probe or 60 oC for RNA probe.

Autoradiography:

  1. The membrane was washed twice for 5-10 min with wash-buffer at room temperature, and twice for 15 min at 65 oC.for with prewarmed (65 oC) wash-buffer.
  2. Remove final wash solution and rinse membrane in 5xSSPE at room temperature. Blot excess liquid and cover in UV-transparent plastic wrap. Do not allow membrane to dry out if it is to be reprobed.
  3. Blot was exposed at -80 oC unsing Kodak XAR film and x-ray intensifying screens.

Reagents and Solutions

  1. 10xE: 0.2M MOPS, 0.05M NaAc and 0.005M EDTA, adjust to pH 7.0 with NaOH;
  2. Sample denaturation mix (100 µl): 64.6 µl Formamide, 22.6 µl Formaldehyde and 13 µl 10xE;
  3. Loading daye mix: 50% Glycerol, 0.3% Xylene cyanol, 0.3% bromophenol blue and 1mM EDTA;
  4. 20xSSPE: 3M NaCl, 0.25M NaH2PO4 and 0.02M EDTA, adjust to pH 7.4;
  5. Pre-hybridization solution: 2.5ml formamide, 1ml 5xP, 1.4ml H2O, 0.292g NaCl and 0.1ml sperm DNA;
  6. 5xP: 0.25M Tris-HCl pH 7.5, 0.5% Sodium pyrophosphate, 1% Polyvinylpyrolidone, 1% Bovine serum albumine, 1% Ficoll and 5% SDS;
  7. Wash-buffer: 1% SDS + 0.1% 20xSSPE.

Northern Blot Protocol

Introduction
Northern blot analysis allows the detection and quantification of specific RNA species from a
particular cell type. Isolated RNA is electrophoresed through an agarose/formaldehyde gel which
separates the RNA species by size. The faster migrating RNA fragments are the smallest,
however, the distance of migration is not linear, rather it is inversely proportional to the size
RNA molecule. When RNA has separated following electrophoresis, it is stained with ethidium
bromide and visualised using ultra violet light. For gels of total RNA the 28S and 18S ribosomal
subunits are visible and act as convenient size markers (approx 4.8 and 1.9kb, respectively).
To probe for a specific mRNA species by northern blot, it is first necessary to transfer the RNA
from the agarose/formaldehyde gel to a nylon membrane. RNA is detected by hybridisation
using a labelled probe. The probe is a DNA or RNA molecule which is chemically or
radioactively labelled. We will use a [32P]-dUTP-labelled probe.
 
Timetable
1st Day Prepare gel and electrophorese
Transfer o/n
2nd Day Synthesize and purify probe
Pre-hybridize membrane & Hybridize with probe o/n
3rd Day Wash membranes and expose to film o/n
4th Day Develop film Ð interpret data
 
Materials required
RNase free water (i.e. DEPC treated) Agarose
10 x running buffer (0.2 M MOPS pH 7.0,
10 mM EDTA)
Formaldehyde
UV transilluminator
Formamide Ethidium bromide
0.5 M iodoacetamide Microfuge
Formaldehyde, Ethidium bromide, UV, High Voltage Ð Caution
Formaldehyde is toxic and a potential carcinogen; use the fume hood Ð do not inhale!
Formamide, ethidium bromide and iodoacetamide are also toxic and should be
handled with gloves
UV light can damage your eyes if not protected Ð use wear facemask or goggles.
Molten agarose can cause nasty burns Ð handle with care.
 
Preparation of the samples
1. Keeping all samples on ice, add 10-20 µg of RNA from the control cells and/or treated cells
to a sterile Eppendorf tube. The volume of RNA should be increased to 15 µl by the addition
of DEPC treated water.
2. To this add 2.5 µl deionised formamide, 2.5 µl formaldehyde and 2.5 µl of running buffer
(10 x stock), and 2.5 µl loading buffer
3. Denature samples by heating at 60ûC for 20 min, then snap cool on ice.
 
Gel Preparation
1. You will be provided with 130 ml molten agarose at 60 ûC (1.5 g agarose/130 ml H20).
2. Carefully add: 15.0 ml 10 x running buffer
3.0 ml 0.5 M iodoacetamide
2.25 ml 40% formaldehyde
10.0 µl Ethidium bromide (10 mg/ml stock)
3. Mix by swirling and pour into sealed gel tray (don’t forget the comb to form the wells).
 
Load and run the gel
1. Load samples into the wells and run at 100 volts for 2 h (e.g. over lunch 
1st Day (PM) Ð stop gel and transfer o/n
Materials required
Hybond N (Nylon) membrane 20 x SSC (0.3M Na3citrate, 3M NaCl)
Saran wrap (DOW chemical company) Glass plate
Whatmann 3MM paper Bottle of water as weight for blot
Sponge soaked in 20 x SSC
Visualise integrity of RNA by UV illiumination/EtBr staining
1. Wearing gloves, remove gel from the tank, carefully place gel on transilluminator and take a
photograph. It is often useful to place a fluorescent ruler on the gel prior to the photograph.
This aids later in matching the position of the ribosomal markers on the photograph (which
may not be actual size) with the final X-ray film of the hybridized mRNA.
2. Use extra blue roll when transporting gel from tank to UV transilluminator. Take care not to
spill the running buffer because it may contain ethidium bromide (EtBr).
 
Transfer to nylon membrane
1. Set up transfer as shown and leave to transfer by capillary action overnight.
2nd Day(AM) Synthesize and purify probe
The Northern blot can be probed with any labelled RNA or DNA. Here we will probe the blot
with DNA which is radiolabelled. See appendix for details of other probes.
Materials required
2 x SSC buffer 50 µCi[a32P]dCTP, 3000Ci/mM
High Prime solution Screw cap vials
Radioactivity Ð Caution
b-particles are emitted by 32P radionucleotides need to be handled with care and
disposed of in a designated radioactive waste bin Ð wear two pairs of gloves
 
Transfer disassembly
 1. Carefully disassemble the transfer apparatus and remove the nylon membrane.
2. Fix the RNA to nylon membranes using UV crosslinker.
3. Label your hybridization chamber
4. Place blot in hybridization chamber (RNA ’side’ facing upwards).
 
Probe synthesis
High Prime is a reaction mixture that contains random oligonucleotides, Klenow polymerase,
labelling grade dATP, dGTP, dTTP, and an optimized reaction buffer concentration in 50%
glycerol for rapid and efficient labeling of DNA with 32P- or 35S-labeled dCTP. The plasmid we
will use contains the P2X2 cDNA. We use this plasmid as the template DNA with High Prime
and [a32P]dCTP.
 
1. Place 25 ng template DNA (final volume 11 µl) in a screw cap vial.
2. Denature DNA by heating in boiling water bath for 10 min.
3. Chill quickly on ice.
4. Pulse spin tube in microcentrifuge.
5. Mix on ice: 25 ng denatured DNA
4 µl High Prime solution
5 µl 50 µCi[a32P]dCTP, 3000 Ci/mM
6. Incubate for 10 min at 37ûC.
7. Stop the reaction by adding 2 µl 0.2 M EDTA and heat to 65ûC for 10 min.
 
2nd Day(PM) Pre-hybridize membrane/hybridize with probe o/n
Materials required:
 
Radiolabelled probe
Hybridisation buffer 50% formamide, 5X SSPE, 5X DenhardtÕs reagent, 1% SDS)
Sonicated salmon sperm DNA (10mg/ml).
 
RadioactivityÐ Caution
Radioactivity should not be used without a demonstrator present.
 
The blot is probed in Northern hybridisation buffer which comprises SDS together with a variety
of reagents to block non-specific background. The probe is double stranded DNA, so it must be
boiled (in order to separate the two strands) before being added to the blot. The probe is applied
overnight in the smallest volume necessary to wet the blot in a shaking water bath or
hybridisation oven.
 
Prehybridise/hybridise
1. Boil sonicated salmon sperm DNA for 5-10min and snap cool on ice. Add to hybridisation
buffer to a final concentration of 200mg/ml. (e.g. 0.3ml of salmon sperm DNA (10mg/ml) to
15ml hybridisation fluid.
2. Add the hybridisation fluid (with added SS DNA) to the hybridisation chamber containing
the membrane. Prehybridise the membraneat 42ûC for at least 1h (no upper time limit).
3. Boil probe for 10 mins and snap cool on ice.
4. Open the hybridisation chamber, and without disturbing the membrane, pour the
hydridisation soulution into a 15-20ml tube (you will not recover the full 15ml). Add
denatured (boiled) probe to the hybridisation buffer and mix gently, then return it to the
hybridisation chamber and incubate overnight at 42ûC.
 
3rd Day(AM) Wash membranes and expose to film o/n
Materials required:
2 x SSC with 0.1% SDS Saran wrap X-ray film
0.1 X SSC with 0.1% SDS
 
The small fraction of the RNA that comprises the P2X2 mRNA on the membrane should have
hybridised to the radioactive probe. However, the membrane will have high background
radioactivity which needs to be washed in order to yield a clean film and clear results.
1. Discard the radioactive hybridisation solution in the designated container/sink. Leave the
membrane in the hybridisation chamber for subsequent washes.
2. Add about 50ml 2 x SSC with 0.1% SDS (room temperature) to the hybridisation chamber
and wash the membrane for 10 min
3. Repeat the above washing step.
4. Discard the second wash solution. Add to the hybridisation chamber, 50ml 0.1 X SSC with
0.1% SDS, preheated to 42°C. Wash for 20 min.
5. Discard the final wash solution. Remove the membrane and air dry of a piece of Whatman
filter paper. Wrap in Saran wrap and expose overnight to X-ray film.
 
4th Day (AM) Develop autoradiograph Ð interpret data
Gels may be developed after 24 hours, or can be left for longer periods if necessary.
Appendix
Saran wrap and gels
Saran wrap is NOT the same as cling film. Saran wrap can be purchased from Sigma. Standard
protocols for these types of gels have high concentrations of formaldehyde that will not allow
ethidium bromide addition so the gel has to be stained afterwards. In this course, we utilise a
lower formaldehyde concentration which means that that ethidium bromide can be included in
the gel. Iodoacetamide is included in the gel as an RNase inhibitor and is hazardous.

Cow System Transgenic Protein Purification System

Introduction to Cow System™: 
The Cow cloning system is an advanced protocol for the purification of 
recombinatn proteins from lactating bovine species. The system is more 
reliable than current sheep cloning methods, requires less starting 
material, is less expensive and will not cause public controversy when the 
press get holds of it. 

1) Description: 
The Cow System™ is the most powerfull system for the cloning and 
expression of recombinat protein. Target genes are cloned in clow plasmids 
under control of the bovine serum albumin promotor. The desired product 
can comprise more than 10% (50kg) of the animal after a few hours of 
fermentation, in inclusion bodies. 

2) Host for cloning: 
Suitable cows for cloning include the Cowalbi, which are albino, and allows 
the black/white screening for the library when plated on the suitable field 
spread with X-Grass™ (GG) selective medium. Note that the Scottish 
Highland Cows (SHC) are more sensitive to the heat shock transformation. 

3) Preparation of the medium: 
You need a very, very big flas (two million liters), a very, very big 
shaker and a clothes peg for your nose. The growth medium, "Cow Dip" or 
C.D. broth, is made from @m grass, 0.5M hay supplement, 0.2mM ice cream 
(vanilla flavour), pH7.2. An oxygen carrier, such as myoglobin, can be 
added to increase growth and prevent drowning. Alternatively, Farmer 
Seah® provide bovine aqualungs for growth in liquid media. Always, 
autoclave the medium in order to kill scrapie prions. 

4) Transformation: 
You need alt least 12kg of plamid, a vet and a big swimming pool at 37C and 
a sauna at 42C. It is possible to use the FarmerKit™ from Farmer 
Seah®. Supercompetent Cowalbi calves are provided with the kit. 
Recently, a protocol derived from Maniatis has been described by Jersey et 
al. (1996) using Brute Force™ on mature bovine. 

a- Place the cows in a room on ice and mix gently to assure that the cows 
are in good shape. 
b- Add 2kg of plasmid directly in each cow using a clean glove. Rotate 
gently to mix. 
c- Place cows on ice for 30 minutes. 
d- Heat shock the cows by placing them in the sauna at 42C for exactly 40 
seconds. Do not vortex; the cows will break the mixer. 
e- Place the cows in a cold room for 2 minutes. 
f- Add two million liters of GG medium. 
g- Incubate the cows at 37C with shaking in Earthquake zone for one hour 
and spread the cows on a green field(*). Note that premade libraries are 
available from Cowtech (Cambridge). 

5) Expression: 
a- To start the culture, add two tonnes of transformed cows (0.25 vol. of 
total bovine prep (TBP)) in the liquid culture (figure 1) 
b- Incubate with shaking the culture at 37C until OD_600 of the supernatant 
reaches 0.8. 
c – The cows could be separated by decantation using a shepherd and his 
dog, by gentle centrifugation (5g) of filtration (filter, 1m). 
d- Lyse by osmotic shock in 2M Chocolate Syrup(**) or with three medium 
shrapnel devices. Atomic weapons are not advised. Note that English cows 
are very resistant to lysis. It is possible to use a French Press. 
e- Purify the product using normal procedures. For enhanced purification, 
use the Cow-Lyse Kit™. 

6) Cloned gene expression in Cow System™ 
The plasmid included with this kit is the new pBSE, the Bovine System 
Expression plasmid. Other Cow-based expression systems can be used but may 
not over-express to the same extent. 
When cloning chimaeric gene constructs, remember to use Cowalbi which are 
missing the gene coding for the restriction enzyme BDNA I. Cowalbi are 
also immunosuppressed to aid expression of hideously deforming genes. 

If gene products are poor, try the Rhino Kit™. This incorporates a 
grey/white selection procedure to replace the Blue Whale Kit™, which has 
been discontinued due to poor yields and international pressures. 

(*) Single cloned cows could be stored as frozen at -80C or in a field in 
Greenland. 
(**) Note that the use of chocolate syrup osmotic shock can lead to 
milkshake production in certain bovine species. 
(***) pMooscript can be used instead for lower expression levels. 
figure 1: Fermentation conditions (Cows floating in a BIG erlenmeyer)