miRNA克隆中的问题，拜托高手了，急～～
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Cloningof miRNAs was performed as described (Elbashir et al., 2001; Llave
et al., 2002a). In brief, ;600 mg of RNA was resolved through two lanes
on a denaturing 15% polyacrylamide gel. Labeled DNA oligonucleotides
were used as size standards. A gel fragment spanning the size range
of 15 to 26 nucleotides was excised, and RNA was eluted overnight with
0.4 M NaCl at 4℃. The RNA was recovered by ethanol precipitation, dephosphorylated,
and again ethanol precipitated. Then, the small RNAs
were ligated sequentially to 5’ and 3’RNA/DNA chimeric oligonucleotide
adapters (Dharmacon Research, Boulder, CO). The 3’ adapter oligonucleotide
(5’-pUUUaaccgcatccttctcx-3’; uppercase, RNA; lowercase,
DNA; p, phosphate; x, inverted deoxythymidine) (Elbashir et al., 2001)
possessing a 5’ monophosphate and a 3’ inverted deoxythymidine to
prevent self ligation, was then ligated to the dephosphorylated small RNA.
The ligation product was recovered from the gel, 5’ phosphorylated, and
the RNA was recovered by ethanol precipitation. Next, the 5’ adapter
(5’tactaatacgactcactAAA; uppercase, RNA; lowercase, DNA) was ligated
to the phosphorylated ligation product as described above.
这是文章里克隆小RNA的方法，我是搞不懂他的3‘和5’街头是怎么设计的，他是用的DNA/RNA杂和的接头，可是给的序列只有一条？？
里面的uppercase是指上面的那条吧，谁能告诉我他的这个接头的设计原理，我知道里面有一个酶切位点！！
谢谢
yongdali@hotmail.com
QQ:153206517
et al., 2002a). In brief, ;600 mg of RNA was resolved through two lanes
on a denaturing 15% polyacrylamide gel. Labeled DNA oligonucleotides
were used as size standards. A gel fragment spanning the size range
of 15 to 26 nucleotides was excised, and RNA was eluted overnight with
0.4 M NaCl at 4℃. The RNA was recovered by ethanol precipitation, dephosphorylated,
and again ethanol precipitated. Then, the small RNAs
were ligated sequentially to 5’ and 3’RNA/DNA chimeric oligonucleotide
adapters (Dharmacon Research, Boulder, CO). The 3’ adapter oligonucleotide
(5’-pUUUaaccgcatccttctcx-3’; uppercase, RNA; lowercase,
DNA; p, phosphate; x, inverted deoxythymidine) (Elbashir et al., 2001)
possessing a 5’ monophosphate and a 3’ inverted deoxythymidine to
prevent self ligation, was then ligated to the dephosphorylated small RNA.
The ligation product was recovered from the gel, 5’ phosphorylated, and
the RNA was recovered by ethanol precipitation. Next, the 5’ adapter
(5’tactaatacgactcactAAA; uppercase, RNA; lowercase, DNA) was ligated
to the phosphorylated ligation product as described above.
这是文章里克隆小RNA的方法，我是搞不懂他的3‘和5’街头是怎么设计的，他是用的DNA/RNA杂和的接头，可是给的序列只有一条？？
里面的uppercase是指上面的那条吧，谁能告诉我他的这个接头的设计原理，我知道里面有一个酶切位点！！
谢谢
yongdali@hotmail.com
QQ:153206517
