Differential Display Protocols

L.L. Espey--01/07/05

OUTLINE OF PROTOCOLS

1. Extraction of total RNA using modified Baylor method (and GenHunter Protocol)

2. Quantitation of extracted total RNA

3. MessageClean step for removal of DNA contamination from total RNA

4. Reverse transcription of mRNA using GenHunter RNAimage Kit

5. PCR amplification of cDNA using GenHunter RNAimage Kit

6. Differential display of amplified cDNA on sequencing gel

7. Remove and dry gel, and expose and develop film for autoradiograph of DD

8. Extract cDNA from the Whatman paper

9. Reamplify the cDNA by PCR

10. Check size of PCR products on agarose minigel

11. Prepare Northern blots for analyzing the uniqueness of the cDNAs

12. Verify the uniqueness of the cDNA by probing a Northern blot

13. Prepare LB-Medium and LB-Agar for cloning

14. Clone the PCR-amplified cDNA, using GenHunter pCR-TRAP Cloning System

15. Prepare a single Tet^R colony containing the plasmid cDNA insert

16. Prepare the clone for sequencing using a Promega Wizard Plus Miniprep Kit

17. Denature the plasmid prior to sequencing, using an Amersham Quick Denaturing Kit

18. Sequence the cDNA insert by using an Amersham Sequenase Version 2.0 Kit

19. Fix the sequencing gel and read the cDNA sequence

20. Use the NCBI database to identify the protein product(s) homologous to the cDNA

NOTE: Bold Bracketed Numbers [X] indicate a convenient stopping place in the protocols.

1. Extraction of total RNA using modified Baylor method

(1) Collect ovarian tissue samples in liquid nitrogen as rapidly as possible.

(2) Before proceeding with extraction, obtain the following materials:

guanidine isothiocyanate (GITC) solution (see next page for formula)

cesium chloride (CsCl) solution (see next page for formula)

250 mL beaker with 0.1 N NaOH for rinsing sonicator

2 X 250 mL beaker with ddH₂O for rinsing sonicator

ultracentrifuge tubes--one per sample (labeled on the side near the bottom)

get dry ice and liquid nigrogen before grinding tissue

(3) Homogenization:

(a) Add 1.0 mL GITC solution to each tube for homogenization.

NOTE: For 6 tubes, prepare 25 mL GITC. Homogenize in 1.0 mL GITC, and add 3.0 mL more GITC and vortex. Layer this 4.0 mL on top of 6.0 mL CsCl₂ in an ultracentrifuge tube.

(b) Rinse sonicator in 250 mL NaOH, and then in ddH₂O for 5-10 sec.

(c) Rinse sonicator in 1.0 mL GITC solution from item "a" above for 5-10 sec.

(d) Add liquid nitrogen to mortar, put in ovaries and grind, then 'spatulate' to homogenizing tube.

NOTE: Add about 0.5 g/mL ovarian tissue to 1.0 mL GITC in homogenizing tube.

(e) Homogenize for 15-30 sec (or longer if homogenate is not homogenous).

(f) Rinse sonicator in ddH₂O, NaOH, ddH₂O, and GITC between samples.

(g) After last sample, rinse sonicator in ddH₂O, then EtOH, and dry with paper towel.

(4) Centrifugation:

(a) Add 3.0 mL more of GITC to 1.0 mL of homogenized sample.

(b) Layer homogenized sample onto 6.0 mL of 5.7 M CsCl solution in ultracentrifuge tube.

(c) Balance each tube against mate, using GITC, and spin (at 30-32K rpm ) at 20°C O/N

(d) Pipet off supernatant with sterile Pasteur pipet to below start of CsCl cushion.

(e) Carefully decant remaining CsCl and let each tube drain on paper towels or Kimwipes.

CAUTION: The RNA pellet might be loose at the bottom of the ultracentrifuge.

(f) Flame a razor blade (held by a hemostat) and use it to cut tubes in two, 1 cm from bottom.

(g) Rinse pellet briefly with 1.0 mL of 70% EtOH at room temperature.

(h) Resuspend pellet in ~300 (or ~200) mL (per gm original tissue) of 1X TE (pH 8.0).

(i) Only if necessary, let samples sit a few minutes at 60-65°C to aid solubilization.

(j) Transfer dissolved RNA to 1.5 mL microfuge tubes.

(5) Extraction:

(a) To RNA soln add 30 (or ~20) mL 3M NaOAc (pH 5.2) + 900 (or ~600) mL cold 100% EtOH.

(b) Shake to mix, and store at -80° C for 15+ min.

(c) Centrifuge at 12,000 g for 10 min at 4° C, and discard the supernatant fluid.

(d) Wash the pellet with 500 mL 70% EtOH, recentrifuge 5 min, decant supnt, and dry tube.

(e) Resuspend RNA in 330/220) mL TE (pH 7.6) and add equal volume SIGMA chisam.

(f) Vortex, bump centrifuge, and pipet 300/200) mL of top layer into a fresh microfuge tube.

(g) Re-ppt with 30/20 mL 3M NaOAc (pH 5.2) and 900/600 mL 100% EtOH at -80°C for 15 min.

(h) Centrifuge at 12,000 g for 10 min at 4° C, and discard the supernatant fluid.

(i) Wash the pellet with 500 mL 70% EtOH, recentrifuge 5 min, decant supnt, and dry tube.

(j) Add 150-300 mL sterile depc H₂O and solubilize at 42-45°C.

(k) Quantitate at 260/280 Å by adding 2 mL of solution to 998 mL dH₂O.

SOLUTIONS FOR EXTRACTION OF RNA

5 M GITC:

59.0 g …..guanidine isothiocyanate

0.242 g….Tris

0.336 g….EDTA

50 mL …..sterile H₂O (DEPC-treated)

Heat to 50-65°C to dissolve GITC, cool, q.s. to 100 mL with sterile H₂O.

Filter through 0.45 mM Nalgene (#130-4045), and decant into sterile bottle.

Add 5 mL B-mercaptoethanol (i.e., 2-mercaptoethanol) prior to use.

5.7 M CsCl:

95.96 g……cesium chloride

1.68 g …….EDTA

50 mL ……..sterile H₂O (DEPC-treated)

Dissolve, and q.s. to 100 mL with sterile H₂O.

Decant into sterile bottle, weigh it, autoclave it, reweigh and adjust volume back to original weight by adding sterile H₂O.

1A. GenHunter Protocol, using RNApure Reagent

A. Extraction:

(1) Put the ovaries for each time interval into a mortar with liquid N₂ , and grind with pestle.

(2) Put ground ovaries into a large tube containing @ 1.0 ml cold RNApure/100 mg ovarian tissue.

(3) Homogenize the tissue for 30-60 seconds, or until the tissue is dispersed.

(4) Allow homogenate to sit on ice for 10 minutes.

(5) Transfer 1 mL aliquots of the homogenate/lysate into 1.5 mL microfuge tubes.

B. Purification:

(1) Add 150 mL of chloroform per mL of homogenate, and vortex for 10 seconds.  (At this point, one can freeze and store the tubes at -80C.)

(2) Spin the tubes in a microcentrifuge at 4C for 10 minutes.

(3) Carefully remove the upper phase, and pool equivalent extracts into a clear 2.0 mL tube.

(4) Centrifuge again, and transfer clear supernatant fluid into a 1.5 mL tube.

(5) Add an equal volume of isopropanol and let sit on ice for 10 minutes.

(6) Mix vigorously by vortexing, and centrifuge for 10 minutes at 4C.

(7) Rinse the RNA pellet with 1 mL of cold 70% ethanol, and centrifuge for 2 minutes at 4C.

(8) Decant off the ethanol, spin briefly, and remove the residual liquid.

(9) Resuspend the RNA in 50 mL of depc-H₂O.  (NOTE:  Do not use SDS in resuspension.)

(10) Heat at 65C for 3-5 min and vortex before quantitating RNA.

(11) Quantitate RNA by mixing 1.0 mL RNA with 1.0 mL H₂O. (Read at 260 nm: 1.00 OD = 40 mg)

(12) Store RNA in aliquots at -80C.

2. Quantitation of extracted total RNA

(1) First, quantitate the stock solution of the total RNA that was extracted. Take 2.0 mL of stock RNA and dilute it to 1.0 mL and read at 260 and 280 nm.

(2) Remember that 1.0 O.D. = 40 mg RNA/mL.

For Example: (Or, simply 20 X O.D. = mg/uL)

At 260 nm: 0-h = 0.574 O.D. = 22.96 mg RNA/1.0 mL of diluted solution

= 22.96 mg RNA/2.0 mL of stock solution

= 11.48 mg RNA/1.0 mL of stock solution

4-h = 0.620 O.D. = 24.80 mg RNA/1.0 mL of diluted solution

= 24.80 mg RNA/2.0 mL of stock solution

= 12.40 mg RNA/1.0 mL of stock solution

At 280 nm: 0-h = 0.343 O.D., and RATIO 260/280 = 1.67

4-h = 0.317 O.D., and RATIO 260/280 = 1.96

(This ratio preferably should be 1.6-1.8 to assure there is not excessive protein in the sample.)

(3) For the MessageClean Step, the total RNA should be at a concentration of 1.0 mg/mL.

(Dilute an appropriate amount of stock RNA to 1.0 mg/mL with depc-H₂0.)

NOTE: Remember that in making dilutions: V₁C₁ = V₂C₂

Where: V₁ = volume (in mL) of stock RNA you will need

C₁ = concentration of stock RNA = x mg/1.0 mL (e.g., 100mL)

V₂ = final volume (in mL) of diluted RNA desired

C₂ = final concentration of diluted RNA = mg/mL

For Example:

0-h: V₁ = (100 mL X 1.0 mg/mL)/11.48 mg/mL

= 8.73 mL (diluted to a total volume of 100 mL)

(Thus, 8.73 mL of stock RNA + 91.27 mL depc-H₂O.)

4-h: V₁ = (100 mL X 1.0 mg/mL)/12.40 mg/mL = 8.06 mL

(Thus, 8.06 mL of stock RNA + 91.94 mL depc-H₂O.)

3. MessageClean step for removal of DNA contamination from total RNA

(1) First digest DNA, using DNase I, adding reagents in the following order:

Reagent mL

total RNA 50.0 (= 50 mg)

10X Reaction Buffer 5.7 (vortex 3-5 sec before adding DNase I)

DNase I (10 units/mL) 1.0

Mix well and incubate for 30 min at 37°C.

(2) UNDER THE HOOD, mix phenol:CHCl₃ in a ratio of 3:1 for extraction to remove proteins.

(Alternatively, use SIGMA chisam in place of the phenol:CHCl₃ mixture.)

(3) Add 40 mL of phenol/CHCl₃ mix to the DNA digest (from step #1, above), vortex for 30 sec, and let sit for 10 min.

(4) Microfuge (at room temp) for 5 min, collect 50 mL supernatant, and transfer to a 500 mL tube.

(5) Add 5.0 mL of 3 M NaOAc and 200 mL 100% EtOH, vortex) and put at -80C for 20 min (or ON).

Prepare EtOH for six extractions (i.e., 0-, 2-, 4-, 8-, 12-, and 24-h) as follows:

95% EtOH = 1900 mL EtOH + 100 mL depc-H₂O = 2000 mL

70% EtOH = 1400 mL EtOH + 600 mL depc-H₂O = 2000 mL

(6) Spin for 10 min at 4°C and pipet off the supernatant.

(7) Wash the RNA pellet with 500 mL of 70% EtOH. (do not disturb pellet)

(8) Spin for 5 min at 4°C and remove the EtOH.

(9) Spin briefly, remove any residual liquid by using fine tipped pipets, and let air dry.

[10] Re-dissolve the RNA in 20 mL of depc-H₂O by 15X pump pipeting.

(11) Take 2mL of the re-dissolved RNA and add 998 mL of dd-H₂O to quantitate at OD₂₆₀.

(NOTE: Store the remaining ~48 mL of re-dissolved RNA at -70°C.)

(NOTE: Determine concentration as follows: (Remember, 1.0 OD₂₆₀ = 40 mg RNA/mL)

Unknown concentration (in mg/mL) = O.D. X 40 mg/mL

For Example:

0-h: if O.D. = 0.1044, then RNA concn = 0.1044 X 40 = 4.176 mg/mL and, since

the re-dissolved RNA was diluted 200X, there are 4.176 mg/5 mL,

or 0.8352 mg/mL.

4-h: if O.D. = 0.0786, then RNA concn = 0.0786 X 40 = 3.144 mg/mL and,

there are 3.144 mg/5 mL, or 0.6288 mg/mL.

(12) Just before RT reaction for DD, dilute RNA to 0.1 mg/mL, using depc-H₂O.

4. Reverse transcription of mRNA using GenHunter RNAimage Kit

(1) After using a "MessageClean Kit" to free the RNA of chromosomal DNA contamination, set up reverse transcription (RT) reactions for each RNA sample in 0.5 mL PCR tubes. (You will be using 1-3 of the different T₁₁ primers for this step.)

(2) Prepare a reaction cocktail as follows:

mL mL X 6.4 mL X 19.2

dH₂0 9.4 60.2 180.0

5X RT buffer 4.0 25.6 76.8

dNTP (250 mM) 1.6 10.2 30.7

TOTAL 15.0 96.0 287.5

(NOTE: If using Gibco RT, you must add 2 mL of DTT soln to cocktail in place of dH₂O).

(3) Add 2.0 mL of one of the appropriate T₁₁ primers to the PCR tube containing cocktail.

(If a number of PCR tubes are to receive a given primer, prepare a "subcocktail".)

(4) Add 2.0 mL of one of the total RNA samples to the PCR tube (of each different primer).

(Dilute appropriate amount of RNA to 0.1 mg/mL with depc-H₂O before using for RT reaction.)

(NOTE: Remember that, in making dilutions: V₁C₁ = V₂C₂)

For Example: If there are 0.84 mg/mL in the 0-h RNA, and you want 20 mL of 0.1 mg/mL:

then, V₁ = (20 mL X 0.1 mg/mL) ¸ 0.84 mg/mL = 2.38 mL

thus, use 2.38 mL of the 0-h RNA plus 17.62 mL of depc-H₂O.

Or: If there are 0.63 mg/mL in the 4-h RNA, and you want 20 mL of 0.1 mg/mL:

then, V₁ = (20 mL X 0.1 mg/mL) ¸ 0.63 mg/mL = 3.17 mL

thus, use 3.17 mL of the 4-h RNA plus 16.83 mL of depc-H₂O.

(5) Program the thermocycler to 65°C for 5 minÞ 37°C for 60 min Þ 75°C for 5 min Þ 4°C.

(The 75°C step inactivates any proteases that might degrade Taq polymerase.)

(6) Add 1 mL MMLV RT to each tube after 10 min at 37°C, and mix quickly by "finger-tipping".

(NOTE: Each time you complete a new RNA extraction (after it is "clean") include a reaction mixture that does not contain reverse transcriptase. By doing this, if there is still DNA contamination after the MessageClean step, then you will see bands on the DD gel. In contrast, if the RNA was clean, then there should be no cDNA present in the RT reaction that excluded reverse transcriptase, and there should be no bands on this “control” lane during the first PCR amplification and DD step.)

(7) At the end of the reverse transcription, spin the tubes briefly to collect condensation.

[8] Set tubes on ice for PCR (next step), or store at -20°C for later use.

5. PCR amplification of cDNA using GenHunter RNAimage Kit

(1) Take primers and buffers out of freezer and put on ice to thaw.

(2) Then, determine the number of reactions, label PCR tubes, and remove isotope from freezer.

(3) Prepare a master mix (i.e., a cocktail) of sufficient quantity for all tubes:

AGENT__________ mL/tube X_6.5 X_13 X_26 __ACTUAL__

dH₂O 9.2 64.0 127.0 254 ____________

dNTP (25 m M) 1.6 10.4 21.0 42 ____________

T₁₁______ 2.0 13.0 26.0 52 ____________

10X PCR buffer* 2.0 13.0 26.0 52 ____________

³⁵S-dATP (1200 Ci/mM) 1.0 3.2 6.5 13 ____________

Taq DNA Polymerase 0.2 1.3 2.6 5.2 ____________

TOTAL MASTER MIX 16.0 104 209 417 ____________

*NOTE: This is Qiagen buffer for Qiagen Taq, and the buffer already contains MgCl₂.

(If using AmpliTaq, use the GenHunter buffer.)

(NOTE: If there is to be a control, with no RT mix, then you must separate mix, here.)

(NOTE: It is important to BE AWARE that some fluid tends to stay in the pipet tips.)

(4) Separate the master mix into 102-104 mL submasters for each AP primer, and add 13 mL of the appropriate AP-primer (2 mM) individually to each submaster tube.

(5) Disperse 18 mL of each submaster into a PCR tube. NOTE: it will be necessary to "bump" centrifuge the submaster tube before pipetting out the last 18 mL aliquot.

(6) Add 2 mL each of the 0-, 2-, 4-, 8-, 12-, and/or 24-hour RT mixes from step #4.8.

(7) Mix well, by pipetting up & down.

(8) PCR at 94°C for 30 sec Þ 38°C for 2 min Þ 72°C for 30 sec (for 40 cycles) Þ 72°C for 5 min Þ 4°C dwell. (Use thermocycler program #69)

[9] Centrifuge for 5 sec, siphon off mineral oil, and retrieve 16-18 mL of amplified product.

(11) Use 3.5 mL of each PCR product plus 2 mL of loading dye for DD on sequencing gel, or store at -20°C for later use.

6. Differential display of amplified cDNA on sequencing gel

(1) Wash glass plates with mild lab detergent (e.g. Alconox), and rinse thoroughly. Dry with a paper towel and then buff plates with kimwipes.

(2) UNDER THE HOOD, siliconize the plates, using SIGMA Silicote. (Unfortunately, different sources give different instructions. Some say to siliconize only the notched plate, and leave the long plate plain. Others say to siliconize both plates. We will only siliconize the notched plate. Wipe the GEL SIDE of the plate with Kimwipe saturated with Silicote, air dry for 5 min, and buff with Kimwipe. (Be sure to keep track of the siliconized side by putting tape on outside.)

(3) Rinse the gel side of both plates with 70% EtOH, and then buff with kimwipes again.

(4) Place the large plate horizontally on a Styrofoam tube rack, put 0.4 mm spacers on each side, place the notched plate on top, and put three clamps on each side of the plates.

(5) Tape the bottom first, with each end of the tape going about 1" beyond the plate corners. Then, put tape on each of the two sides, and re-apply clamps before pouring the gel.

(6) Prepare 6% acrylamide sequencing gel according to kit instructions, or by following formula:

(NOTE: Acrylamide is a neurotoxin--always wear gloves.)

(a) Urea: measure out 24.0 g ultrapure urea.

(b) 30% acrylamide stock: 28.5 g acrylamide + 1.5 g bis-acrylamide, to 100 mL with dH₂0.

(c) 10X TBE: Use a mix to prepare a stock soln of 10X TBE. (Add 178 mL dH₂O to stock jar).

(d) 10% APS stock: 1.0 g ammonium persulfate + 10 mL dH₂O.

(e) TEMED: an electrophoresis purity grade of this agent.

MIX: 24.0 g urea + 16.0 mL dH₂O + 5.0 mL TBE stock + 10.0 mL acrylamide stock

(Heat to 40°C to dissolve, add dH₂O to 50 mL total volume, then cool to 25°C. This usually requires heating for 20-22 seconds in the microwave.)

(Discard, and start over, if temperature goes above 50°C.)

ADD: 400 mL APS, and 20 mL TEMED

(Swirl solution vigorously and pour gel immediately.)

(7) Insert the straight side of the comb into top of gel up to first edge of oval holes.

(8) Let plates sit horizontally for at least 2 hours. (If left overnight, cover ends with 1X TBE Kimwipes, and wrap with Saran wrap.)

(9) Remove tape from bottom, place plates in system, add 1X TBE to reservoirs, remove comb, rinse space where comb was located, and re-insert comb with teeth down, 0.5 mm into gel.

(10) Warm up at 70 watts and 32 mAMP (and maximum voltage) until gel reaches 45-50°C.

(11) Take 3.5 mL sample plus 2 mL loading dye and heat at 80°C for 2 min before loading on gel.

(12) Rinse wells, load, and run at 50 watts/23 mAMP until xylene is 10-15 cm from bottom of plates.

(NOTE: Voltage should not be significantly above 1,700 volts.)

7. Remove and dry gel, and expose and develop film for autoradiograph of DD

(1) Turn on the gel dryer to 80°C.

(2) Remove plates from sequencing system, and place on a Styrofoam tube rack to remove tape.

(3) Place plates on flat surface, and with notched plate on top, gently pry plates apart.

(4) Place a properly sized piece of 3M paper over the exposed gel, and press it with a roller.

(Be sure to mark the upper-left corner of the paper to show upper-left corner of gel and date.)

(5) Slowly peel the paper off the large, bottom plate, so that the gel adheres to the paper.

(6) Turn the 3M paper over (to expose the gel) on a flat surface and cover the gel with Saran wrap.

(7) Insert the 3M paper, with gel face up, in the gel dryer on top of 1-2 pieces of 3M paper.

(8) Dry the gel for 60 minutes, and then remove the Saran wrap before exposing to X-ray film.

(9) In the darkroom, place the gel against a piece of X-ray film, and staple twice on each side.

(Use Amersham ßMAX HyperFilm, or Kodak X-OMAT AR film) (Staple top/bottom for 2nd exposure, and corners for 3rd exp.)

(10) Expose the film to the gel for 15-48 hours at -80°C. (cold temp is optional)

(11) Remove the staples, and develop the X-ray film according to Kodak instructions:

(NOTE: Be sure exposed side of film is up in pan, and developer completely covers film.)

(a) KODAK GBX Developer: 3-4 min (no agitation) (200 mL Stock + 724 mL H₂O)

(b) KODAK GBX Fixer: 3-4 min (agitation) (200 mL Stock + 800 mL H₂O)

(c) Water Wash/Rinse: 10 min (clean tap water)

(12) Allow film to air dry, date it, and then label the different lanes, immediately.

8. Extract cDNA from the Whatman paper

(1) After developing the film, use hypodermic needles to orient the autoradiograph with the gel by lining up the stapler holes. (NOTE: Stick the needles through to a cork.)

(2) Locate the bands of interest on the film, and poke the four corners of each band with a hypodermic needle (to identify the desired area on the gel).

(3) Use a sterile scalpel blade (or, razor) to cut the desired bands from the gel.

(4) Soak the gel slice in 60 mL dH₂O in a siliconized microfuge tube for 5-10 min.

(5) Place in thermocycler at 100C for 10-15 minutes.

(6) Centrifuge for 2 min to collect condensation and pellet the gel and paper debris.

[7] Store the resuspended cDNA at -20°C, or use 4 mL for re-amplification in the next step.

9. Reamplify the cDNA by PCR

(1) Take the primers and buffers out of freezer to thaw.

(2) Re-amplification should be done using the same primer set(s) and PCR conditions, except the dNTP concn is 20 mM (i.e., use 250 mM dNTP stock) instead of 2-4 mM, and no ³⁵S.

(3) Prepare a master mix (i.e., a cocktail) of sufficient quantity for all tubes:

AGENT_________ mL/tube_ X_6.5 X_8.7 X_10.9 X_13.0 ACTUAL

dH₂O 20.4 133 178 226 265 _________

dNTP (250 m M) 3.2 21 28 35 42 _________

T₁₁_____ 4.0 26 35 44 52 _________

10X PCR buffer* 4.0 26 35 44 52 _________

Taq DNA Polymerase 0.4 2.6 3.5 4.4 5.2 _________

TOTAL MASTER MIX 32.0 208 280 349 417 _________

*NOTE: This mix is based on using Promega/Quigen Taq, and buffer containing MgCl₂.

(4) Next, separate master mix into individual PCR tubes in 32 mL aliquots.

(5) Add 4 mL of the appropriate AP-primer (2 mM) individually to each PCR tube (or to master mix).

(6) Add 4 mL each of anyone of the 0-, 2-, 4-, 8-, 12-, and/or 24-hour cDNA extracts from step #8.13.

(7) Mix well, by pipetting up & down.

(8) Hot-start at 94°C for 2 min Þ PCR at 94°C for 30 sec Þ 40°C for 2 min Þ 72°C for 30 sec (for 40 cycles) Þ 72°C for 5 min Þ 4°C dwell. (Use thermocycler program #67)

[9] No step, here, just relax!

[10] Use 5 mL of each PCR product plus 1 mL of loading dye for electrophoresis on agarose gel, and/or store unused product at -20°C for later use.

(NOTE: 10 mL of remaining PCR product can be used for the Northern in Step #12, below.)

10. Check size of the PCR products on agarose minigel

(1) Prepare 2% agarose gel by dissolving 1.0 g agarose (USB Cat.#32829 for low M.W.) in 50 mL of 0.5X TBE by bringing to a brief boil (95-98°C) over a flame (or, in a microwave).

(2) Allow the agarose mixture to cool to 60-65°C and immediately add 5 mL of ethidium bromide. (NOTE: The ethidium bromide stock solution should be at a concentration of 10 mg/mL.)

(3) Place the gel apparatus on a flat surface, and center the balancing bubble to level it.

(4) Pour the agarose mixture into a small minigel apparatus to the 1 cm mark, remove any trapped air bubbles, insert the large-toothed comb (12 wells), and let the gel set for 40-60 min.

(5) Carefully remove the comb by gently pulling it straight up while holding down the gel holder.

(6) Carefully remove both rubber end-blocks and re-position the gel in the chamber with the sample wells oriented toward the negative electrode.

(NOTE: Be careful not to let the gel slide out of the tray.)

(7) Slowly fill chamber with 0.5X TBE buffer until top surface of gel is submerged approx 2 mm.

(8) Add 1 mL of loading dye to 5 mL of ladder (ladder = 50, 150, 300, 500, 750, 1000 bp).

(9) Add 1 mL of loading dye to 5 mL of the cDNA samples (from Step #9.10).

(10) Load all the samples onto the gel.

(11) Carefully place the safety cover onto the chamber, ensuring that both banana plugs are secure, and connect the leads to the power supply.

(12) Initially run the power supply for ~5 min at ~90 volts, until the samples enter the gel. Then, increase the voltage to ~130 volts.

(13) Run the gel for ~30 min (don't let orange loading dye reach the positive end of the agarose gel).

(14) Turn off the power supply, and disconnect the leads.

(15) Observe the gel for cDNA bands by examining the gel under UV light with protective glasses. (NOTE: The UV transparent gel tray has a fluorescent ruler on the under side.)

[16] Take a picture of the gel in order to have a record of the bands.

(NOTE: If the gel shows a weak band, or no band, dilute first-round PCR sample (Step #9.10) 1:10 and use 4 mL of this as a template for another 40-cycle amplification (i.e., repeat Step #9).

11. Prepare Northern blots for analyzing the uniqueness of the cDNAs

NOTE: Take the formamide out of the freezer to thaw.

NOTE: Set a heat block at 65°C.