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Unidirectional Cloning - Krizman Protocol 2
Either micro-bulk or microdissected tissues can be the starting material
for the cDNA library protocol described below. If you have any technical questions, please
contact Dr. David Krizman who developed the method at NCI.
| Part A - First Strand Synthesis |
| 1.
|
Primers Needed:
| Primer | Sequence |
| 5' PCR primer | 5'- CUA CUA CUA CUA TAC GGC TGC GAG AAG ACG ACA GAA -3' |
| 5' SWITCH oligonucleotide | 5'- TAC GGC TGC GAG AAG ACG ACA GAA GGG -3' |
| 3' CDS primer | 5'- GGA TCG CTC GAC ATC GAT ACG AC(T) 30VN-3'
(V=A,G, or C); (N=A, G, C, or T |
| 3' CAU primer | 5'-CAU CAU CAU CAU GAT CGC TCG ACA TCG ATA CGA C-3' |
|
| 2.
|
Combine the following reagents in a sterile 0.5 ml microcentrifuge tube:
3µl RNA/H
20 mixture
1 µl 10 µM 5' SWITCH oligonucleotide
1 µl 500 ng/µl 3' CDS oligonucleotide
»Total reaction volume = 5 µl
|
| 3.
|
Mix contents of the tube and spin down briefly.
|
| 4.
|
Incubate the tube at 65°C for 5 min.
|
| 5.
|
Place tube directly at 42°C for 5 min.
|
| 6.
|
Mix the following reagents together and preheat in tube at 42°C for 2 min.
2.0µl 5X first-strand buffer
1.0 µl 20mM DTT
1.0 µl 10mM dNTP mix
1.0 µl Superscript II (200 units/µl, Life Technologies Inc.)
»Total reaction volume = 5.0 µl
|
| 7.
|
Add the pre-heated reagents to the RNA reaction tube. Mix tube by pipetting.
|
| 8.
|
Incubate RT reaction at 42°C for 30 min.
|
| 9.
|
Place reaction on ice to terminate the first strand synthesis. Store at -20°C.
|
| Part B - PCR Amplification |
| 1.
|
Mix the following reagents in a 0.5 ml microcentrifuge tube.
5 µl first strand cDNA (save the remaining 5 µl)
77 µl sterile, deionized H2O
10 µl 10X PCR buffer (Boehringer Mannhiem)
2 µl 10mM dNTP mix
1 µl 50 µM 5' PCR primer
1 µl 50 µM 3' CAU primer
1 µl PerfectMatch (Stratagene)
»Total reaction volume = 97 µl |
| 2.
|
Dilute 1.0 µl (5 units) Taq DNA polymerase (Boehringer Mannheim) in 2.0
µl of sterile, deionized H20.
|
| 3.
|
Place reaction in the thermalcycler and preheat to 94°C for 3 minutes.
|
| 4.
|
Cool reaction to 80°C for 1 minute. |
| 5.
|
Add 3 µl of diluted Taq and proceed with amplification:
| #Cycles
|
Temperature °C
|
Time
|
| 25
|
94
68
72
|
15 sec
15 sec
3 min
|
| 1
|
72
|
5 min
|
|
| 6.
|
Maintain at 4°C
Notes:
| a) | Always use a negative PCR amplification control that
consists of the above reagents with no cDNA template. |
| b) | The use of HotStart is absolutely necessary to reduce the amount of non-specific amplification of single stranded
cDNA by the PCR primers. |
|
| 7.
|
Analyze 10 µl each of the library, (+), and (-) control PCR on
a 1.2% agarose/EtBr gel.
|
Purify PCR product in a Chroma Spin+TE 400 (CLONTECH cat.# Kl323-2) size selection column.
| 1. | Resuspend the column
resin by shaking. Centrifuge the column at 2000 RPM for 5 min to remove the suspension buffer. |
| 2. | Add 10 µl H20 to the library PCR to bring the
volume to 100 µl. Replace the collection vial and add the 100 µl PCR
reaction to the top of the packed resin column. Centrifuge at 2000 RPM for 5 min and collect the follow-through
in a fresh collection tube. |
| 3. | Add the 100 µl volume to a fresh Chroma Spin+TE 400 and repeat the column purification step
as described. Collect the second flow-through in a fresh collection tube. Add 50 µl of a PEG (30% PEG-8000,
30 mM MgCl2) solution and centrifuge at 14,000 rpm for 15 minutes at room temperature. Wash pellet with 70% EtOH,
air dry, and resuspend in 10 µl H20 |
| Part D - Subcloning of cDNA Library |
Reagents are from the CloneAmp cloning kit from Life Technologies (Cat.# 18381-012).
|
1. | Set up the UDG cloning reaction on ice by adding the
following reagents:
1 µl purified cDNA
5 µl sterile H2O
1 µl 10X PCR buffer (Perkin-Elmer)
2 µl pAMP1
1 µl uracil deglycosylase
»Total reaction volume = 10µl |
|
2. | Incubate the reaction at 37°C for 30 min. |
|
3. | Store at 4°C. |
|
4. | Perform standard bacterial transformation. |
|
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