Small RNA (ribonucleic acid) tags
A small molecule and labeling technology, which is applied in DNA/RNA fragments, DNA preparation, recombinant DNA technology, etc., can solve the problems of wasting sequencing resources, affecting sequencing throughput, and inability to mix and sequence Solexa small molecule RNA library samples, etc., to reduce Sequencing cost, effect of increasing sequencing throughput
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Embodiment 1
[0168] Method one method of building a library:
[0169] 1) Take 10ug of mouse total RNA, mix it with an equal volume of 2×gelloading dye (containing formamide), denature at 65°C for 5 minutes, and then place it on ice. The samples were then added to the wells of 15% denaturing PAGE gel. At the same time, take 1 μL of 10bp DNA ladder and 2 μL of 14-30ssRNA ladder marker and mix them with 6×loading buffer respectively, and add them to the spotting wells for electrophoresis.
[0170] 2) After electrophoresis, small RNAs of 18-30 nt were recovered, and 400 μL of 0.3 M NaCl was added to the recovered crushed gel. Placed on a mixer, and eluted at room temperature for 4 hours. Transfer all the gel pieces and eluent in the tube to the Spin-X tube, centrifuge at 14,000 rpm for 2 minutes at room temperature, and discard the broken gel. Add 3 μL glycogen, 1000-1500 μL 100% ethanol (the volume of ethanol added is 2.5-3 times that of the eluent, if the amount of eluent increases, the a...
Embodiment 2
[0210]Use method 2 to build a database:
[0211] 1) Take 5ug of mouse liver total RNA, mix it with an equal volume of 2×gel loading dye, denature at 65°C for 5 minutes, and then place it on ice. The samples were then added to the wells of 15% denatured PAGE gel. At the same time, take 1 μL of 10bp DNA ladder and 2 μL of 14-30ssRNA laddermarker and mix them with 6×loading buffer and add them to the sample wells for electrophoresis.
[0212] 2) After electrophoresis, small RNAs of 18-30 nt were recovered. Add 400 μL of 0.3M NaCl to the gel containing the recovered crushed gel. Placed on a mixer, and eluted at room temperature for 4 hours. Transfer all the gel pieces and eluent in the tube to the Spin-X tube, centrifuge at 14,000 rpm for 2 minutes at room temperature, and discard the broken gel. Add 3 μL glycogen, 1000-1500 μL 100% ethanol (the volume of ethanol added is 2.5-3 times that of the eluent, mix well and store at -80°C for 1 hour. Centrifuge at 14000 rpm at 4°C for...
Embodiment 3
[0251] Use method 2 to build a database:
[0252] 1) Take 5ug of mouse liver total RNA, mix it with an equal volume of 2×gel loading dye, denature at 65°C for 5 minutes, and then place it on ice. The samples were then added to the wells of 15% denatured PAGE gel. At the same time, take 1 μL of 10bp DNA ladder and 2 μL of 14-30ssRNA ladder marker and mix them with 6×loading buffer and add them to the wells for electrophoresis.
[0253] 2) After electrophoresis, small RNAs of 18-30 nt were recovered. Add 400 μL of 0.3M NaCl to the gel containing the recovered crushed gel. Placed on a mixer, and eluted at room temperature for 4 hours. Transfer all the gel pieces and eluent in the tube to the Spin-X tube, centrifuge at 14,000 rpm for 2 minutes at room temperature, and discard the broken gel. Add 3 μL glycogen, 1000-1500 μL 100% ethanol (the volume of ethanol added is 2.5-3 times that of the eluent, mix well and store at -80°C for 1 hour. Centrifuge at 14000 rpm at 4°C for 30 m...
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