Filename:LIBMAKE.doc Written by: M.Schiller (7/4/97)
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Protocol: cDNA Library Production
1.USE SILICONIZED TUBES FOR ALL PROCEDURES.
2.ALWAYS PPT DNA AT -20 oC, not -80 oC
3.ALWAYS USE ENZYMES FROM TWO MANUFACTURERS WHEN POSSIBLE
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Synthesize First Strand Of cDNA
From cell lines or tissues make at least 6 ug poly A+ RNA. For cell line use 5-6 plates of
80% confluent monolayer cells. First make total RNA using stat-60 (see protocol RNApur) then purify the poly A+ RNA using magnetic beads (see protocol PolyA). Quantitate
a small portion of poly A+ RNA using spectrophotometer. Discard measured sample.
Check the RNA by Northern blot with a known positive control probe (preferably a large
transcript) to ensure no degradation of RNA.
Precipitate the 6 µg poly A+ RNA by adding 4 µl of 5 mg/ml glycogen (ambion), 1/10
volume of 3.0 NaAc RNase free, pH 5.2, and 3 volumes of ice cold ethanol. and
precipitate at -20 oC overnight. Spin for 15 min, remove supernate, add 1.0 ml 70%
ethanol, spin for another 5 min, remove supernate, and air dry in fume hood for 10 min.
Resuspend pellet in 20 µl of DEPC H2O. All may not resuspend so make sure DNA does
not dry too long.
Heat RNA to 65 oC for 5 min. cool to room temp for 5 min.
Add 2 µl of 100 mM CH3HgOH, incubate 1 min at RT.
(NOTE: caution (CH3)2Hg this is extremely toxic and can kill at low levels)
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Add 4 µl 700 mM -mercaptoethanol (190 µl H2O + 10 µl stock ME)
(Final concentrations are 10 mM CH3HgOH; 110 mM ME).
Add 4 µl H2O to 30 µl total volume
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Synthesize First Strand Of cDNA as follow using stratagene reagents:
(For each library do two reactions)
Reagent
cold
hot
10X first strand buffer ....................................5 µl .............................2 µl
1st strand CH3-NTPmix .................................3 µl .............................1.2 µl
Linker primer .................................................2 µl .............................0.8 µl
DEPC water ...................................................9 µl .............................5 µl
RNase inhibitor ..............................................1 µl .............................1 µl
[32P] -dCTP(3000Ci/mMol;10mCi/ml) .........0..................................3 µl after anneal
Mix, then add denatured RNA
incubate 10 min at RT for annealing..............25 µl ...........................5 µl
add superscript RT (200 U/µl) .......................4 µl .............................1.6 µl
MMLN RT .....................................................1 µl .............................0.4 µl
incubate at 40 oC for 1 h
Eipper/Mains Protocol Manual
Filename:LIBMAKE.doc Written by: M.Schiller (7/4/97)
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Analysis Of cDNA Synthesis
Before proceeding, analyze cDNA synthesis using PEI strips as follows:
PEI strips are 5 x 1 cm
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Remove 2 µl from the [32P] reaction and add 5 µl 0.2 M EDTA and 15 µl TE buffer.
Spot 4 µl on PEI strip 1 cm from bottom. Save the remainder of the sample for an
agarose gel.
Add 1 M HCl, 1 M NaCl to a beaker covering 0.5 cm of the bottom. After PEI strip
dries, add so that spot is just above liquid and let capillary action do its thing until the
liquid is just short of the top of the strip.
Cut the strip in thirds and count each sample in the scintillation counter. Typical CPM
are top=2,000; Bottom =22,000; middle = 550,000. The bottom is the labeled cDNA
which in this case represents about 4% incorporation. Since the nucleotides are 10 mM
and the volume is 50 µl. Hence 0.5 µmole of nucleotide were incorporated into the
cDNA. Assuming an average size of 2.5 kB cDNA, 0.2 nMol of cDNA strands were
synthesized. Thus, 3 x 1013 molecules of cDNA were synthesized.
Second Strand Synthesis
Synthesize second strand as follows
Reagent
Cold
Hot
Volume...........................................................50µl ............................18µl
2nd Strand buffer ...........................................20µl ............................10µl
2nd Strand nucleotides ...................................6µl ..............................3µl
DEPC water ...................................................111µl ..........................62.5µl
Total ............................................................187µl ..........................93.5µl
cool reaction to less than 16 C.
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Add:
RNase H .........................................................2µl ..............................1µl
DNA polymnerase I .......................................11µl ............................5µl
Incubate at 37 oC for 2.5 h
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Remove 5 µl of reaction, add 5 µl 0.2 M EDTA and 12 µl TE buffer. Spot 4 µl on PEI
strip 1 cm from bottom, run and analyze PEI stips as above. Save the remainder of the
sample for an agarose gel.
Extract reaction 1 X with Phenol:chloroform:isoamyl alcohol (200 µl) and pool the non
radioactive and radioactive samples.
Extract reaction 1 X with Phenol:chloroform:isoamyl alcohol(400 µl)
Extract reaction 1 X with chloroform:isoamyl alcohol(400µl)
Add 40 µl 3.0 M NaAc, mix, add 1.0 ml ethanol, mix and store at -20 C overnight.
Eipper/Mains Protocol Manual
Filename:LIBMAKE.doc Written by: M.Schiller (7/4/97)
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Analysis Of First And Second Strand Synthesis By Agarose Gel
Pour a giant gel 250 ml TAE 1% agarose. Run rest of the aliquot from 1st and 2nd strand
synthesis reactions at 110 V until nucleotide is 1 cm from bottom. Cut off the bottom
100 bp and toss. Dry gel and expose to film 6-14 hours. Should have ds cDNA run upto
at least 10 kB and ss cDNA all the way to the top.
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Generate Blunt Ends On cDNA
Spin down cDNA for 30 min, wash with 80% ethanol, air dry 5-10 min.
Dissolve in 39.5 µl water
dd: 5 µl 10X T4 DNA polymnerase buffer, 2.5 µl dNTP mix (stratagene), 3 µl T4 DNA
polymerase (3U/µl, NEB). Incubate at 16 oC for 30 min.
dd 350 µl TE
Phenol extract with phenol:chloroform:IAA with 400 µl 2X as above.
Phenol extract with chloroform:IAA with 400 µl 1X as above.
Add 40 µl 3.0 M NaAc pH 5.2, and 1.0 ml cold ethanol and precipitate at -20 oC for
several hours or preferably ON.
Spin 30 min, wash pellet with 70% ethanol, and air dry 10 min.
Resuspend pellet in 9 µl of EcoR1 adapters.
dd 1 µl of 10X ligase buffer, 1 µl of 10 mM ATP When DNA completely dissolves add 1
µl (4 U/µl) T4 DNA ligase, mix spin down and incubate at 8 C overnight.
In morning add 1 µl rATP, 1 µl ligase and incubate overnight.
Optional: Ty never does! Heat inactivate ligase at 70 C for 30 min.
Spin 5 sec, cool to RT 5 min, and add 290 µl TE.
Extract 2X with phenol:chloroform:IAA (300 µl) and 1x with chloroform:IAA (300 µl).
Precipitate DNA by adding 30 µl of 3.0 M NaAc, and 1.0 ml ethanol at -20 C.
Pellet DNA 30 min, rinse with 80% ethanol , spin 5 min, air dry 5 min (10 min is hard to
resuspend)
Phosphorylate DNA as follows:
Resuspend DNA pellet in
30 µl water
4 µl 10x kinase buffer (NEB)
4 µl 10 mM rATP (stratagene)
1 µl T4 polynucleotide kinase (10U/µl)
Incubate at 37 C for 30 min, spike with 1 µl T4 polynucleotide kinase and incubate
another 30 min at 37 C.
Add 170 µl TE (pH 7.5). repeat phenol extractions, and ethanol ppt (steps 14-16 above).
Resuspend pellet in 42 µl H2O , 5µl 10X universal buffer (stratagene), and 3 µl Xho1 (40
U/µl). After 45 min incubation add 1 µl Xho1 (Boehringer Mannhiem 10 U/µl), and
incubate an additional 1.5 hours at 37 C.
Add 150 µl TE, pH 7.5, extract with phenol:chloroform and ethanol ppt overnight (steps
14-16 above).
In morning, spin down DNA 30 min, wash with 80% ethanol, spin 5 min, airdry 5 min
and resuspend in 30 µl 1X running buffer clonetech and 5 µl Xylene cyanol/bromophenol
blue 10X sample buffer.
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Eipper/Mains Protocol Manual
Filename:LIBMAKE.doc Written by: M.Schiller (7/4/97)
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Size fractionation of DNA using Clonetech columns
Get bubbles out of Clonetech column (F0565f or K105-1), run column dry, rinse 2X with
1X running buffer.
Run dry, add sample, run dry, rinse tube with 70 µl running buffer, add to the column and
run dry.
Add 100 µl of the column, run dry. Start collecting 1 drop fractions. Add 600 µl 1X
running buffer to the column and collect 25 1-drop fractions. Chrenkov count each
sample. Remove 3 µl of peak samples for gel.
Pool all samples upto peak fraction. Save samples after peak fraction.
Extract 2X with phenol:chloroform:IAA (200 µl) and 1x with chloroform:IAA (200 µl).
Ppt DNA by adding 18 µl of 3.0 M NaAc, pH 5.2, 4 µl 5 mg/ml glycogen, and 0.5 ml
ethanol at -20 oC overnight.
Pellet DNA 30 min, rinse with 1.0 ml 80% ethanol , spin 5 min, air dry 5 min, and
resuspend in 20 l H2O. Run 4 µl on gel to check size and recovery (optional)
Ligate Arms On cDNA As Follows
Add 4 µl H2O, 2 µl vector arms (1 µg/2µl), 10 µl cDNA, 2 µl 10X ligase buffer (NEB), 1
µl T4 DNA ligase (high conc, 400,000 U/µl NEB), 1 µl 10 mM rATP. Incubate at 12 C
overnight.
(Note: Ligase buffer cannot contain peg)
Remove 10 µl to package, add 0.5 µl 10 mM rATP, and 0.5 µl ligase and ligate at 12 C
overnight.
Package Phage DNA
To package half the ligation reaction. Thaw 2 Gigapack III extracts from dry ice rapidly,
and add 5 µl of ligated cDNA/tube. Gently pipette to mix, spin briefly
Incubate for 105 45 min.
Add 500 µl PSM buffer, 20 µl chloroform, mix, spin 10 sec at 14,000 rpm to separate
phases. Remove and store sup at 4 oC.
Before Packaging Other Half Of Library, Check Titer
Make serial dilutions of 10 µl of the packaged phage and plate dilutions of 102-106 as
described in lib-titr.wp.
Calculate the titer. Typical titers of the primary library should be greater than 106 total
phage.
If titer is successful package the rest of the ligated cDNA and process as above. Combine
samples. To 1.0 ml library add 75 µl DMSO, aliquot into 3 tubes and store at -80 C.
Library Amplification
Amplify 1o library as follows:
20 to 40 LB plates (at least 2 days old) are plated with phage at a concentration of 75,000
phage per plate. Grow all day and leave at RT ON. The phage should be close to or lytic
in the morning.
Add 5 ml PSM to each plate, cover all plate and mix in the cold occasionally during the
day.
Eipper/Mains Protocol Manual
Filename:LIBMAKE.doc Written by: M.Schiller (7/4/97)
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Remove PSM from the plates and pool. Add another 5.0 ml to each plate and incubate in
the cold ON.
In the AM remove and pool all PSM collected. Add chloroform at 1 ml/25 ml of library,
mix and spin 5 min. Save sup.
Add 75 µl DMSO /ml of library, aliquot and store at -80 C
Titer amplified library. Titers should be 109-1011 pfu/ml.
Chemical
-mercaptoethanol
CH3HgOH
Glycogen
ZAP kit
Vendor
Ambion
Stratagene
Solutions:
Special notes, common problems, and troubleshooting:
References:
Eipper/Mains Protocol Manual
Catalog #