Department of Biology
Memorial University of Newfoundland
cDNA Library Synthesis
Equipment:
Ice bucket
Microcentrifuge
Micropipetters
Water bath 72 C
Water bath 42 C
Thermocycler
Starting Material:
3 ul (or less) total RNA (0.05-1 ug)
Reagents and disposable Materials:
0.5 ml microcentrifuge tubes
Micropipette tips
Oligonucleotide
3pdT primer
5X First strand cDNA synthesis buffer
DTT (20 mM)
dNTP (10 mM)
Reverse Transcriptase
deionized water
10X PCR buffer
50X dNTP
5prime PCR primer
3prime-dT primer
First strand cDNA synthesis
1) Combine the following in a 0.5 ml microcentrifuge tube:
3 ul total RNA (0.05-1 ug)
1 ul oligonucleotide
1 ul 3prime-dT primer
2) Mix and briefly spin to place sample at tube bottom.
3) Incubate at 72 C for 2 minutes
4) Chill on ice for 2 minutes.
5) Mix and briefly spin to place sample at tube bottom.
6) Add the following in each reaction tube:
2 ul 5X First strand cDNA synthesis buffer
1 ul DTT (20 mM)
1 ul dNTP (10 mM)
1 ul Reverse Transcriptase
7) Mix by gentle micropipetting and briefly spin to place sample at
tube bottom.
8) Incubate at 42 C for one hour.
9) Place tube on ice to terminate first strand cDNA synthesis.
10) Place 2 ul of reaction in chilled 0.5 tube for next step and store
remainder at -20 C for up to three months.
cDNA synthesis by PCR
1) Combine the following in a 0.5 ml microcentrifuge tube:
2 ul First strand cDNA
80 ul deionized water
10 ul 10X PCR buffer
2 ul 50X dNTP
2 ul 5prime PCR primer
2 ul 3prime-dT primer
2 ul polymerase mix
2) Mix and briefly spin to place sample at tube bottom.
3) Cap and place in preheated thermocycler.
a) 95 C for 20 seconds
b) 25 cycles of :
95 C 5 seconds
68 C for 6 minutes
c) 4 C hold to finish.
Go Home!
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Day 2
Equipment:
Ice bucket
Microcentrifuge
Micropipetters
Water bath 72 C
Water bath 45 C
Starting Material:
amplified cDNA
Reagents and disposable Materials:
1.5 ml microcentrifuge tubes
Micropipette tips
deionized water
Taq polymerase
100 mM dATP
proteinase K
Tris EDTA buffered phenol
24 chloroform:1 isoamyl alcohol
isopropanol
70% ethanol
salt solution
topoisomerase-treated vector
chemically competent E. coli
Luria Broth
10% X-gal
LB plus ampicillin agar plates
37 C incubator (plus shaking platform)
cDNA dA Tailing
1) To ensure dA tail addition, recover the amplified cDNA and add:
1 ul of Taq polymerase
1 ul of 100 mM dATP
and incubate at 72 C for 15 minutes.
Proteinase K digestion
1) Combine the following in a 1.5 ml microcentrifuge tube:
50 ul of amplified cDNA
2 ul proteinase K
(store remaining amplified cDNA at-20 C for up to 3 months)
2) Mix and briefly spin to place sample at tube bottom.
3) Incubate at 45 C for 20 minutes.
4) Add 950 ul deionized water.
5) Add 250 ul of Tris EDTA buffered phenol and 250 ul of 24 chloroform:1
isoamyl alcohol.
6) Mix by gentle inversion for 1 to 2 minutes.
7) Centrifuge at room temperature for 10 minutes @ 12K to separate
the phases.
8) Add 600 ul of room temperature isopropanol to a fresh sterile 1.5
ml microcentrifuge tube and add the top aqueous layer.
9) Centrifuge at room temperature for 20 minutes @ 12K to precipitate
the DNA.
10) Remove the supernatant and carefully wash the pellet twice by the
careful addition of 500 ul of 70% ethanol followed by a brief spin and
removal of the liquid by micropipette. After the second wash, centrifuge
for 10 seconds and remove the remaining liquid by micropipette.
11) Let air dry for 10 minutes.
12) Dissolve in 10 ul deionized water to produce concentrated amplified
cDNA.
Ligation and Transformation
1) Combine the following in a 1.5 ml microcentrifuge tube:
10 ul of concentrated amplified cDNA
2ul of salt solution
1 ul topoisomerase-treated vector
2) Incubate at room temperature for 20 - 30 minutes.
3) Add to thawed chemically competent E. coli cells and gently mix.
Do not micropipette to mix.
4) Incubate on ice for 10 minutes.
5) Heat shock for 60 -90 seconds at 42 C and replace on ice to recover
for 1 minute.
6) Add 500 ul of Luria Broth and incubate at 37 C with shaking for
30 -40 minutes.
7) Add 10 ul of 10% X-gal.
8) Plate 250 ul onto 2 LB plus ampicillin agar plates.
9) Incubate at 37 C overnight.
email me at bestave@mun.ca