------------------------------------------------------ 150ul Total
b. c. d. e. f. g. h. i.
briefly vortex mix, spin, remove content into 1.5ml Nuclease-free tube 16C, 2h
put the tube on ice, add 2ul T4 DNA polymerase(5U/ul) briefly vortex mix, spin 16C, 5 min place the tube on ice
add 10ul 0.5M EDTA pH8, to stop the reaction (optional) briefly vortex mix, spin
5.2.3 Phenol chloroform (Corrosive) isoamyl alcohol (PCI) and EtOH precipitation
NOTE: Extraction should be done in hood wearing safety goggles. Waste should be disposed of in a separate designated container.
Important Safety NOTE: Phenol is a corrosive agent which burns skin upon contact, practice extreme safety when performing the following steps.
a. b. c. d. e. f.
spin two Eppendorf phase-lock Light tubes (Green)
remove cDNA reaction content to phase-lock tube, transfer tubes to fume-hood. (lab#459) add 150ul PCI/tube, mix by shaking the tube vigorously for 15 second centrifuge @ 13000g RT, 5min
transfer top layer into 1.5ml clean Nuclease-free tube for each tube, add the following component:
15ul (1/10vol) 1.2ul
3M NaOAc, PH5.2
Glycogen (to help precipitate DNA)
EtOH
415ul (2.5vol)
g. h. i. j.
mix by inverting the tubes.
place the tubes in -70C for >1h (optional).
centrifuge @ 13000g RT for 20min. (prepare 2% agarose gel while waiting).
remove tubes from centrifuge gently, look for a white ~1 mm size pellet on the hinge side. This pellet should be present even if there were no DNA/RNA. If not present, check to see if all reagents were added. Spin again if needed to see the pellet.
k. l. m. n. o.
remove the supernatant with tip pointing away from the pellet, check to make sure the pellet remains after you remove the supernatant wash pellet with 500ul 70% EtOH centrifuge @ 13000g RT for 2min.
remove the supernatant, air dry @RT for 10min or SpeedVac dry for 5min
resuspend pellets of 2 tubes in total 15ul of nuclease-free H2O (sample can be stored @ -20C).
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5.2.4 Gel separation
a. b. c. d. e. f.
prepare 2% 1XTAE agarose gel 0.1 mg/ml Ethidium Bromide (Carcinogen) (need ~30min) save 2ul of sample (as a control) and run gel-purified sample together on Bioanalyzer add 2.5ul 5x loading dye (Qiagen) to remaining sample
pipette mix and load on gel, load 50bp & 100bp DNA ladder (Fermentas)
run gel @ 100V for ~10 min (to let the sample migrate from well in to gel quickly) then @50V for ~30min (cm to maximize separation of small molecular weight primer away from the cDNA)
image gel for 1 second (to minimize UV exposure) and save photos of both pre-cut and post-cut gels. cDNA are normally not visible @1 second exposure time, gel picture can be adjusted later so that cDNA are visible. The primer should run below 50bp. g. h.
cut gel slice from 100bp up to well using UV transluminator (make sure not to include the primer into your gel piece), place it in a 15 or 50ml falcon tube
Important! Minimize UV exposure to yourself (use long sleeves lab coats and face shield) and to the samples (use laminated paper to block UV to the samples not being cut). Turn off UV after cutting each sample. Transfer agar pieces to tubes with normal light. Review cut gel to make sure the right pieces of gel were transferred. i.
take another gel picture with 5 second exposure time. clean the gel and image area (gel slice can be stored @-20C)
j.
5.2.5 Gel extraction
a. b. c. d. e. f. g. h. i. j. k. l. m. n. o. p. q.
weight the gel slice add 3X volume of QC buffer
rotate the tube @37C or 50C incubator for 15 or 10min respectively, until no visible gel slice present add 1X volume of isopropanol, mix
the following steps can be performed by vacuum or centrifuge (13000g, 1min per) load the sample to Minelute column
applied the column to vacuum manifold or centrifuge, repeat this step 3-4 times if you have more than 750ul (load sample to another column if you have more than 3ml) apply 500ul QG buffer for each column, vacuum or centrifuge
apply 750ul PE buffer for each column, vacuum or centrifuge, repeat this step once place the column into emptied 2ml tube, centrifuge @13000g, 1min rotate tube 180C, centrifuge @13000g, 30second place the column to a nuclease-free 1.5ml tube add 12ul EB, wait 1min centrifuge @13000g, 1min.
use same EB solution to elute the other tube of the same sample (sample can then be stored @ -20C) determine cDNA concentration by Bioanalyzer (Bioanalyzer 7500 kit). cDNA should be >80ng/ul (total >1ug) if you start with 2ug PolyA RNA.
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5.2.6 Sonication
a. b. c. d. e. f. g. h. i. j.
use pump to circulate ice water to cool sonicator to 4C. dilute >1ug cDNA in 100ul TE in a 1.5 microfuge tube.
place tube in a rack in water bath that allows the bottom of the tube to touch the sonicator horn. make sure the liquid level of the sample is below the sonicator bath.
sonicate for 8 min at 1 min intervals with 1 min off between to allow sample to cool. spin 10sec.
Minelute purify cDNA, elute in 12ul
determine DNA concentration and size by Bioanalyzer DNA1000 kit. cDNA should be >80ng/ul (total >1ug) range 100-800bp.
gel purify cDNA between 100 to 800bp if needed (see step 4, 5 for details)
5.3 454 library preparation
*GS FLX Titanium General Library Preparation Kit, etc.
5.3.1 Fragmentation of RNA
a. b. c. d. e. f. g. h. i. j. k. l. m. n.
Start with 200 ng of sample RNA in a 200 μl PCR tube. Add Molecular Biology Grade Water to a fi nal volume of 19 μl.
Pipet 1 μl of this solution into a new 200 μl tube, and add 2 μl of Molecular Biology Grade Water. This sample will be compared to the fragmented RNA later on.
To the 18 μl of sample RNA, add 2 μl of RNA Fragmentation Solution, and vortex. Spin the tube for 2 seconds in a mini centrifuge.
Preheat the thermocycler to 70°C before placing the sample tube in. Heat the tube at 70°C for 30 seconds, with the heated lid on. Immediately, place the tube on ice.
Add 2 μl of 0.5 M EDTA, pH 8.0 and 28 μl of 10 mM Tris HCl, pH 7.5. Vortex and spin for 2 seconds in a mini centrifuge.
Add 80 μl of RNAClean reagent, containing SPRI beads.
Mix well by pipetting up and down 10 times, and incubate at room temperature for 10 minutes. Place the tube on a 96 ring Magnetic Particle Concentrator (MPC).
When the beads have completely pelleted on the side of the tube, carefully remove and discard all supernatant, without disturbing the beads.
Keeping the tube on the MPC, wash the beads three times, as follows, without disturbing the beads: i. ii. o. p. q. r.
Add 200 μl of 70% ethanol.
Completely remove and discard the ethanol.
Keeping the tube on the MPC, uncap the tube and air dry the pellet at room temperature for 3 minutes. Remove the tube from the MPC. Add 19 μl of 10 mM Tris.HCl, pH 7.5.
Vortex for 20 sec, spin for 2 seconds in a mini centrifuge, and pellet the beads on the MPC.
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s. t. u. v.
Without disturbing the beads, transfer the supernatant (~19 μl), containing the RNA, to a new 200 μl PCR tube. Place the tube on ice.
Pipet 1 μl of this solution in a 200 μl tube, and add 2 μl of Molecular Biology Grade Water. This sample will be compared to the one from step 3.
Run 1 μl of the fragmented (from step 19) and 1 μl of the non-fragmented RNA (from step 3) on an RNA 6000 Pico Chip on the Agilent 2100 Bioanalyzer to confi rm that the material has been fragmented Proceed with the fragmented sample (~17 μl).
5.3.2 Double-stranded cDNA Synthesis
Retrieve a cDNA Synthesis System Kit (Roche) and thaw the reagents before placing them on ice. Also thaw a tube of Roche Primer “random” 400 μM. 5.3.2.1 Denaturation of RNA a. b. c. d.
To the fragmented RNA, add 4 μl of the 400 μM Roche Primer “random”. Vortex for 10 seconds.
Spin for 2 seconds in a mini centrifuge.
Incubate the tube at 70°C for 10 minutes and immediately after, place on ice for 2 minutes.
5.3.2.2 First Strand cDNA Synthesis a.
With the tube on ice, add the following reagents: 8 μl vial 1 (5× RT-buffer AMV) 4 μl vial 3 (DTT, 0.1 M) 4 μl vial 7 (dNTPs, 10 mM)
1 μl vial 4 (Protector RNase Inhibitor, 25 U/μl) 2 μl vial 2 (AMV RT, 25 U/μl) 40 μl Total volume b. c. a.
Vortex gently for 2 seconds and spin for 2 seconds in a mini centrifuge.
Incubate the tube at 25°C for 10 minutes and then at 42°C for 60 minutes, and transfer on ice. To the 40 μl of sample, add the following reagents: 30 μl vial 9 (5× 2nd strand synthesis buffer) 72 μl vial 12 (redist water) 1.5 μl vial 7 (dNTPs, 10 mM)
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5.3.2.3 Second Strand cDNA Synthesis
6.5 ul vial 10 (2nd strand enzyme) 150 ul Total volume b. c. d. e. f.
Vortex 5 seconds and spin for 2 seconds in a mini centrifuge. Incubate the tube at 16°C for 2 hours.
Add 20 μl of vial 11 (T4 DNA Polymerase) and vortex gently for 5 seconds. Incubate at 16°C for 5 minutes.
Add 17 μl of 0.2 M EDTA, pH 8 to stop the reaction. Vortex 5 seconds and spin for 2 seconds in a mini centrifuge.
5.3.2.4 Double-stranded cDNA Purifi cation a. b. c. d. e. f.
Transfer the sample to a new 1.7 ml microcentrifuge tube. Add 300 μl of AMPure beads.
Vortex for 10 seconds and incubate at room temperature for 5 minutes. Place the tube on a Magnetic Particle Concentrator (MPC).
When the beads have completely pelleted on the side of the tube, carefully remove and discard all supernatant, without disturbing the beads.
Keeping the tube on the MPC, wash the beads three times, as follows, without disturbing the beads: a) b) g. h. i. j. k. l.
Add 800 μl of 70% ethanol.
Completely remove and discard the ethanol.
Keeping the tube on the MPC, uncap the tube and air dry the pellet at room temperature for 3 minutes. Remove the tube from the MPC.
Add 16 μl of 10 mM Tris-HCl, pH 7.5. Vortex for 20 seconds and spin for 2 seconds in a mini centrifuge. Place the tube on the MPC, wait for the beads to pellet on the wall of the tube and transfer the SUPERNATANT
(~16 μl), containing the double-stranded cDNA to a new 200 μl PCR tube, and place the tube on ice. We recommend processing the cDNA sample through to the end of the protocol. However, the cDNA sample can be stored over night at -80°C.
5.3.3 Fragment End Repair
Obtain a Rapid Library Prep kit. a.
In a 1.7 ml microcentrifuge tube, prepare the End Repair mix, as follows.
2.5 μl RL 10× Buffer 2.5 μl RL ATP 1 μl RL dNTP 1 μl RL T4 Polymerase 1 μl RL PNK
1 μl RL Taq Polymerase
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