miRNA cloning from Arabidopsis thaliana
(Junjie Li, 01/02)
Oligo Sequence:
Chimeric oligos (P-Phosphate; idT-Inverted deoxythymidine)
Rp4: 5’- dA.dT.dC.dG.dT.A.G.G.C.A.C.C.U.G.A.A.A
Rp5: 5’- P.U.U.U.dC.dT.dG.dT.dA.dG.dG.dC.dA.dC.dC.dA.dT.dC.dA.dA.dT.idT
RT-PCR primers
Microp1:
5’- ATCGTAGGCACCTGAAA
Microp2:
5’- ATTGATGGTGCCTACAG
Total RNA extraction
-Use mixed-tissue Ler plant (inflorescence, leaves, stems) by Tri-Reagent protocol
-Isolate silique total RNA separately
-Combine above together
Isolation of small RNA from total RNA
32P End-labeling:
Oligo
-32PATP (10uCi/ul, 6uCi/pmolATP)
10xBuffer
T4 PNK (10U/ul)
ddH2O
Total
-37oC 30min
-65oC 20min to inactivate
1ul (10pmol)
3ul (5pmol)
3ul
2ul
21ul
30ul
Electrophoresis
-Load sample: total RNA (~600ug), 32P-labelled RNA size marker
-Resuspend RNA in denaturing loading buffer
-Loading buffer:
Formamide
80%
1XTBE
Bromophenol Blue(BPB)
1mg/mL
Xylene Cyanol FF
1mg/mL(optional)
-Make-up:
Formamide 40ml
5xTBE
10ml
BPB
50mg
Cyanol
50mg
Total
50mL
-15% polyacrylamide, 8M urea gel
Gel recipe (for 16cmx16cmx1.5mm gel): (modified from Kiledjian lab)
Urea
19.2g(8M)
5xTBE
8mL
30% Acr:Bis (29:1) 20mL
APS (10%)
320ul
TEMED
24ul
H2O
Total
40mL
Running gel:
-Remove comb and wash wells with syringe to clean free urea
-Pre-run for 40min
-Load samples, run gel
-Stop running, remove one of glass plates
-cover with plastic wrap and put on fluorescence markers to locate this gel position
-Expose gel to film and develop it
-find expected size range (in plants, 18-28 nt) and cut corresponding gel band and crush
-Add 1-2volumes of RNA elution buffer
RNA elution buffer recipe (from Kiledjian Lab):
20mM Tris(7.5)
0.5M NaAcetate
10 mM EDTA
1% SDS
Buffer make-up: 1M Tris(7.5)
1ml
3M NaAc(no PH)
8.3ml
0.5M EDTA
1ml
20% SDS
2.5 ml
Total
50ml
-Rt 12 hrs or 65c 4 hrs
-Go through glass wool
-Chloroform extraction twice
-2.5 volumes Ethanol-precipitate with 20ug glycogen as carrier, 10 min at –20oC
-Spin 10 min at top speed
-Wash in 70% ethanol
-Air-dry 5min
-Resuspend in DEPC-treated water
3’-adaptor(Rp5) ligation
Dephosphorylation of small RNA
Small RNA
10xNE Buffer 3
CIP (10U/ul)
Total
-37 oC for 60 min
-phenol/chloroform extraction
-ethanol precipitation with glycogen
25 ul
3 ul
2 ul
30 ul
Ligation
Rxn:
ddH2O(DEPC)
10xBuffer (BSA free)
0.1% BSA
Adaptor (100pmol/ul)
Small RNA
T4 RNA ligase (40U/ul)
3 ul
3 ul
13ul
10 ul
1 ul
Total 30 ul
5 oC for 18 hours
Purification of small RNA with 3’-adaptor
15% sequencing gel
RNA elution from gel
5’-adaptor ligation
5’-phosphorylation:
Rxn:
small RNA-3’adaptor(Rp5)
10xPNK Buffer
T4 PNK (10U/ul)
ATP (cold, 5nmol/ul)
DdH2O (DEPC)
32 ul
5 ul
3 ul
10 ul (1mM final)
Total 50 ul
-37 oC for 30 min
-65 oC for 20 min
-phenol/chloroform extraction 1x
-chloroform extraction 2x
-ethanol precipitation with 1ul Glycogen (20ug/ul)
5’-adaptor(Rp4) ligation:
Rxn:
ddH2O(DEPC)
10xBuffer (BSA free)
0.1% BSA
Adaptor (Rp4, 100pmol/ul)
Small RNA-Rp5
T4 RNA ligase (40U/ul)
2.5 ul
2.5 ul
10 ul
10 ul
1 ul
Total 25 ul
5 oC for 18 hours
Purification of small RNA with 5’- and 3’-adaptor
15% sequencing gel
Elution and purification
Reverse transcription reaction and amplification
RT Rxn:
DEPC H2O
3.5 ul
Small RNA with adaptor
Microp2 (10uM)
-Mix, 70 oC for 10 min, on ice for 1 min
-10x MuLV RT Buffer
2 ul
-10 mM dNTP
1 ul
-40U/ul RNase inhibitor
0.5 ul
-Mix, 42 oC for 1 min
-25 U/ul MuLV Reverse Transcriptase
2 ul
-42 oC for 1 hour
10 ul
1 ul
PCR amplification:
RT products
10xBuffer
dNTP (1mM)
Microp1
Microp2
ddH2O
Taq
4 ul
5 ul
2.5 ul
1 ul
1 ul
38.5 ul
1 ul
Total 50 ul
DNA cloning
-Phenol/chloroform extraction 1x
-Chloroform extraction 2x
-Ethanol precipitation with 1ul Glycogen (20ug/ul)
BanI Digestion:
PCR products
10xBuffer (4)
BanI (20U/ul)
DdH2O
37 oC overnight (~20 hours)
Concatamerization: Small oligo
10xT4 ligase Buffer
T4 DNA ligase
10 ul
5 ul
5 ul
30 ul
12 ul
1.5 ul
1.5 ul
Total 15 ul
-RT or 16 oC
-Purification by 4% low melt agrose gel, cut size ranging from 200 bp to 800 bp
Taq polymerase treatment:
Concatamerized fragments(200-800bp)
10xBuffer
dNTP (2.5mM)
x-Taq
72 oC for 20 min
TOPO TA cloning (PCR2.1 vector, invitrogen)
8 ul
1 ul
1 ul
o.1ul
Fresh Taq-treated products
Salt solution
TOPO vector
Perform according to the manufacture
4 ul
1 ul
1 ul
Sequencing
Pick out plasmids with insert >200 bp and do sequencing