T4 DNA ligase ligates RNA molecules
T4 DNA Ligase joins double-stranded complexes, including RNA-DNA heteroduplexes and RNA-RNA heteroduplexes, defectively.
Operation method
Ligating RNA molecules with T4 DNA ligase
Materials and Instruments
T4DNA Ligase T4 Polyribonucleic Acid Kinase RNA Donor Receptor Oligonucleotide cDNA Template Move I Materials and equipment Caveat For effective ligation, the concentration of the bridge cDNA template is very important, preferably equal to or between the concentrations of the two RNA molecules to be ligated; if there is too much template, the RNA molecules will bind to a different cDNA template, but not to the other RNA molecule. We recommend that the concentration of RNA in the ligation reaction should preferably be between 0.1 and 10umol/L. For more product details, please visit Aladdin Scientific website.
10X Ligation Buffer [Y32P]ATP RNase-free Water l0 mmol LATP RNasin. RNase-free DNase. 5XTBE 2X Denaturing Gel Sampling Buffer TE
Vacuum Centrifuge Concentrator
1) T4DNA ligase
2)T4 polyribonucleic acid kinase
3)RNA donor
4) Receptor
5) Oligonucleotide cDNA template
6) 10X ligation buffer: 500 mmol/LTris-HCl(pH7.5), 100 mmol/LMgCl2, 200 mmol/LDTT,0.5 mg/mL BSA(for NewEngtandBiolabs ligase) or 660 mmol/LTris-HCl(pH7.6), 66 mmol/LMgCl2,lOOmmol/LDTT(for Amersham/USB ligase). mmol/LMgCl2,lOOmmol/LDTT (for Amersham/USB ligase)
7)[Y 32 P]ATP (10mCi/ml,300Ci/mmol=3.33umol/L)
8) No RNase Water
9) l0 mmol/LATP
10)RNasin.
11) DNase without RNase.
12) Vacuum centrifuge concentrator.
13) 5XTBE:54 gTris, 27.5 g boric acid, 20 ml0.5mol/LEDTA (pH8.0)
14) 2X Denaturing Gel Sampling Buffer: 10 mol/L urea, 1XTBE or 80% formaldehyde, 1XTBE
15) TE; 10 mmol/L Tris-HCl(pH8.0),lmmol/LEDTA
II. Methods of operation
1. Phosphorylation of 3' RNA
1) Add 5ul of [γ32P]ATP (50ulCi,16pmol) to a 1.5 ml tube and dry it in a vacuum centrifuge concentrator.
2) Add 0.5ul of 10X ligation buffer, 3ul of 20umol/L 3'RNA, 1ul of water, and 0.5ul of T4 polynucleotide kinase to this tube, and the final volume is 5.0ul.
3) Incubate at 37℃ for 30~60 min.
4) Incubate at 75°C for l0min or 92-95°C for 2 min to inactivate the polynucleotide kinase. Place on ice
2. Ligation reaction
1) In the inactivated polynucleotide kinase reaction tube, add 1.0ul of 10× ligation buffer, 3.0ul of 20umol/L 5'RNA, and 3ul of 20umol/L cDXA template.
2) Heat to 75℃ for 2 min and leave at room temperature for 5 min to hybridize RNA and DNA.
3) Add 1.l0 mmol/L cold ATP, 2ul T4DNA ligase to the hybridization reaction solution to a final volume of 15ul.
4) Incubate at 30°C for 2~4 hrs.
5) Add lul of RNase-free DNase, incubate at 37℃ for 15~30 min.
6) Add 16ul of 2X Sampling Buffer and electrophoresis the sample to detect the labeled 5' RNA and 3' RNA as well as the full-length molecules that are equivalent to the ligated products.