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RNA ISOLATION
Arruffo and Seed recommend a version of the GTC/PsCl method which calls for banding in a SW55 rotor for two hours. Given that I have not tried this technique, the rotor is not available in our lab, and they have not used it with tissue I have not included it here. RNA isolated by the standard GTC/CsCl method works very well.
POLY A+ SELECTION
This is a variation on the method we use in the lab. I found it to give high quality poly A+ and to be a little less tedious than the method we use.
1. All materials should be RNAse-free. In addition, to increase your yield I use a siliconized syringe for the column.
2. You need a bed volume of 0.3 ml of packed oligo dT cellulose per milligram of RNA used. There is no advantage to using a larger column and you are probably increasing the losses. If you are using less than a .3 ml column you can use a 1 ml syringe. Otherwise increase to a 3 ml syringe.
3. Prepare the column (with a GT/C filter at the bottom) by .washing with 5M NAOH and then rinsing with RNAse free water.
4. Prepare oligo dT by measuring out 0. 5 ml dry volume of the
resin into an appendorf. Add 1 ml O.lM NAOH. Let equilibrate approx. 5 mins. Pour into the column the amount needed to achieve the desired bed volume. Rinse with H20 until pH of eluate is neutral. Rinse with 2-3 mls. of loading buffer. Then remove resin to a 15 ml. blue cap tube (be careful to discard the GT/C filter) and add 4-6 mls. loading buffer.
5. Heat total RNA to 700C for 2-3 mins. Add LiCl (from a 2M stock) to make concentration 0.5 M.
6. Add RNA to oligo dT mixture. Vortex or agitate on rocker for 10 mins. at room temperature. The idea is to keep the oligo dT suspended in the RNA mixture to allow the RNA to bind.
7. Pipet the mixture back into the column (with new filter in bottom) and allow the buffer to elute.
8. Run 3 ml of loading buffer through. Then 3 ml. of middle wash buffer, allow column to run dry. Check the O.D. 260 of the last 300 ul. If less than .025 go on to number 9. Otherwise wash with another 1-2 mls. middle wash buffer. This elutes the poly A- RNA from the column.
9. Wet sterile Q-tip with water. wipe out top of column above resin if using a 3 ml column.
10. Elute poly A+ RNA directly into a SW50 tube. Use 1.5 mls. elution buffer for this.
11. Add 150 microliters 3M NaOAc to tube and fill to top with
ETOH. Cover with parafilm and mix. Place at -200C for at least 30 mins. I often do overnight.
12. Pellet RNA at 50K for 30 mins. Pour out ethanol mixture. Let tube air dry. Resuspend in 50-100 ul water (depending on how much RNA was loaded). Take 2 ul dilute into 200 ul water and check the O.D. 260 to determine the yield. You can also
run 1 microgram of RNA on gel to check that it has not been degraded.
BUFFERS
LiCl - 2 M stock
Loading Buffer - 0.5 M LiCl, 2 mM EDTA, 0.1% SDS, 10 mM Tris pH 7.5
Middle Wash Buffer - 0.15 M LiCli 2 mM EDTA, 0.1% SDS, 10 mM Tris pH 7.5
Elution Buffer - 2 mM EDTA, 0.1% SDS 5N NAOH and 0.1 NAOH
Oligo dT cellulose
CDNA SYNTHESIS
This is a variation of the Gubler and Hof fman procedure. Reverse transcriptase is used to make the first strand by priming from oligo dT. The second strand is made using only Polymerase I and RNAse H. Given the high concentrations of Pol I in this protocol, T4 polymerase is not required to blunt the ends and is not recommended by the authors. Again, all buffers need to be RNAse free. Use siliconized eppendorf tubes.
1. Use 3-5 ug of poly A+ RNA in water.
2. Heat to 1000C for 30 seconds - quench on ice.
3. Adjust volume to 70 ul with water.
4. Add 20 ul RT1 buffer.
5. Add 2 ul RNAsin (Promega or BMB).
6. Add 5 ug of oligo dT primer
7. 2 ul of 25 mM dNTP's (Pharmacia, of US Biochemicals)
8. 1 ul of 1 M DTT
9. 100 units of Rev. Transcriptase
10. Incubate 420C for 1 hour.
11. Heat to 700C for 10 mins. to inactivate RNAse Inhibitor and RT.
SECOND STRAND SYNTHESIS
1. Dilute above reaction mixture with 320 ul water.
2. Add 80 ul RT2 buffer
3. Add 5 ul BMB Polymerase I (5 units/ul).
4. 2 ul RNAse H (BRL, 2 units/ul).
5. Incubate at 150C for 1 hour.
6. Incubate at 220C for 1 hour.
7. Add 20 ul 0.5 M EDTA
8. Phenol:Chloroform extract x 1.
9. Add 10 ug linear acrylamide
10. Add 50 ul 5 M NaCl
11. Add 1 ml ETOH - mix
12. Spin at room temperature for 4 minutes. vortex briefly, and spin another 2 mins.
13. Carefully remove supernatant - these pellets are not sticky.
14. Rinse with 70% ETOH. Spin briefly after removal of ETOH and remove last traces of ETOH.
15. DO NOT DRY PELLET.
BUFFERS
Linear acrylamide - it is an inert carrier which helps with the precipitation of DNA and RNA. It is cheap and does not appear to interfere with ligation or other enzymatic steps. It is made by making a standard 5% acrylamide solution without any bis, adding APS and temed and letting it polymerize at room temperature. I have made enough for the lab to last until the year 2000. Use as a 5 ug/ul stock solution.
RT1 Buffer 0.25 M Tris-pH 8.8 (8.2 at 420C), 0.25 M KC1, 30 mm
MgCl2
RT2 Buffer 0.1 M Tris-pH 7.5, 25 mM MgCl2, 0.05 M KC1, 0.25
mg/ml BSA (Acetylated - nuclease free), 50 mM DTT
5 M NaCl
Phenol/CHC13
ETOH
PREPARATION OF LINKERS
The next step is to ligate on the adaptors to obtain a CACA overhang on each end of the CDNA. The linkers can be bought from invitrogen or made by a DNA synthesizer. If they are made in the lab, prepare them in the following way.
1. Make on column with Trityl group OFF.
2. Bake in Ammonium hydroxide for 5 hrs.
3. Dry down in speed-vac.
4. Resuspend in water at a concentration of 1 ug/ul.
5. Add MgSO4 to a concentration of 10 mM.
6. Add 5 volumes of ETOH.
7. Spin ten minutes in centrifuge.
8. Rinse with 70% ETOH.
9. Resuspend in TE to a concentration of 1 ug/ul.
10. Kinase adaptor by taking 25 ugs oligomer, add 3 ul lOx kinase buffer, and 20 units kinase.
11. Place at 370C overnight.
LINKER LIGATION
1. Resuspend CDNA in 240 ul TE
2. Add 30 ul lOx ligase buffer.
3. Add 7 ul commercial linker OR 3 ul kinased 12-mer and 2 ul kinased 8 mer.
4. Add 2 ul, NEB T4 Ligase.
5. Add 5 ul, 5 mg/ml BSA.
6. Place at 150C overnight.
10 x Ligase Buffer
.5 M Tris-HCl (pH 7.8)
100 MM MgCl2
200 mM DTT
10 MM ATP
500 ug/ml BSA
SIZE FRACTIONATION
This step accomplishes two goals, (1) to get rid of small (presumably incomplete) oDNAs in order to increase the quality of the library and (2) to get rid of the extra linkers.
1. Take ligated CDNA mixture, phenol extract with equal volume. You may need to back extract the phenol phase with 100 ul H20 to increase recovery. Combine the two aqueous pools.
2. Add 50 ul 5 M NaCl to CDNA. Mix
3. Fill eppendorf tube with ETOH. Mix
4. Spin 4 minutes, vortex lightly, spin 2 minutes.
5. Remove supernatant.
6. Resuspend with 100 ul TE.
The original protocol called for pouring a continuous
potassium acetate linear gradient. Given the scarcity of tubing for the gradient maker I chose to use a step gradient. If a linear gradient maker is available, use itl
1. Make up four solutions. All are 2 mM EDTA, 1 ug/ml Ethidium Bromide. They are 20%, 15%, 10%, and 5% in KOAC. You should make a 40% KOAC stock and filter sterilize that.
2. Use a SW50 polyallomer tube.
3. Pour two gradients (for balance).
4. Place 1.2 mls of each solution in the tube starting from the bottom with 20%. Carefully layer in the lighter ones so that you do not get excess mixing.
5. Pipet the CDNA mixture on top of the gradient. Balance with water for the other one. Weigh to make sure they are equal.
6. Spin 50 K for three hours (no longer).
7. Drip out fractions into siliconized eppendorf tubes. Do this by using a butterfly needle with attached tubing. (Clip off the leuer lock). Pierce tube near the bottom. Get a clamp to clamp off tubing if needed.
8. Collect 3 - 0.5 ml fractions. Then 9 -0.25 ml fractions. Discard the rest.
9. Add 5 ug linear acrylamide to each sample. Fill tube with ETOH.
10. Chill at -200C at least 4 hours if not overnight.
11. Spin out 10 minutes at room temperature. Pellet should be visible.
12. Remove supernatant. Rinse with 70% ETOH.
13. Spin out last drops, but DO NOT DRY.
14. Resuspend each fraction in 20 ul TE.
15. Run .7% agarose gel with size markers and 2 ul from each sample.
16. Pool the fractions that are the sizes you want. This is your Tinkered CDNA.
PREPARATION OF VECTOR
The vector for this library is described in the papers by Arruffo and Seed. In order to prepare it for making the library you need to remove a 350 bp fragment that is flanked by two inverted (with respect to each other) BstXl sites. This is where the CDNA will be ligated in. In order to get a library with an acceptable background it is crucial to completely cut the vector. The cut vector is then isolated from the fragment by running a preparative gradient.
Cut the vector in appropriate restriction buffer, plus 50 ug/ml BSA overnight at 500C. Use a large excess of BstXl.
The next day pour KOAC gradients exactly as described in the CDNA fractionation section. Load the restricted mixture 10 ug/gradient on top of the gradient. Centrifuge at 50 K for 3 hrs., 220C. Look at the gradients under a UV light. You should see a band approximately two thirds of the way down, pull it out with a syringe, add linear acrylamide and three volumes of ETOH to precipitate. Resuspend in 50 ul TE.
LIGATION OF CDNA TO VECTOR
In this step you create recombinant clones. It is necessary to ligate different concentrations of CDNA and vector in order to find the optimal ratio. If you add too much vector you will increase your background unnecessarily, if you use too much CDNA you will decrease the titer because if two different cDNAs bind to the same vector it can't be ligated closed and is lost.
I use approximately .05 ug vector in each ligation with four different concentrations of CDNA, .2 ul, .5 ul, 1 ul, 2 ul and the fifth sample is vector alone.
Do these in 10 ul legations (i.e., 1 ul lOx ligase, buffer H20 to equal 10) . Dilute T4 ligase 1:10 in appropriate diluent, and use 1 ul per reaction.
Ligate at 150C at least three hours.
To work-up f or electroporation you need to have the DNA in water. If you are going to transform using standard methods then you can transform directly from the ligation.
I w/u the ligation by adding 40 ul water, + 50 ul phenol/CHC13, extract. Transfer the aqueous phase to a new tube, add 50 ul water, 66 ul 5 M ammonium acetate, 5 ug linear
acrylamide and 350 ul ethanol. mix, spin 3 mins., vortex, spin 2
minutes as above. Rinse with 70% ethanol. Then dry the DNA.
Resuspend in 4 ul distilled water. For electroporation you must
get rid of all the salt and all the ethanol.
ELECTROPORATION
This is based on the protocol developed by Bio-Rad. it achieves 1-5 x 109 transformants/ug reproducibly. The protocol to make competent cells is included at the end of this set of protocols.
To do the electroporation itself: it is important to keep everything on ice during this @ eriment (cuvette, cuvette holder, etc., and to immediately resuspend the cells in SOC after shocking).
1. Thaw prepared cells on ice.
2. Add 2 ul DNA/40 ul cells. The DNA concentration is several orders of magnitude below saturation.
3. Let sit on ice 2 min.
4. Add to .2 cm cuvette.
5. Shock at 25 UF, 800 ohms, 2.5 kEV. Time constant should come out around 20.
6. Immediately add 1 ml SOC to the cuvette and pipet up and down.
7. Let cells shake at 370C for one hour.
8. Plate out 100 ul and 100 ul of a 1:20 dilution.
9. Figure out the best ratio of vector to CDNA to give you low background and highest titer.
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