| 查看: 740 | 回复: 4 | |||
[交流]
求助英译汉
|
|
Cloning and sequencing of chicken miRNAs. Secondary CEFs, prepared by routine techniques, were infected with the RB1B strain of MDV for 24 h at a multiplicity of infection of 25,000 PFU/106 cells. Protocols developed previously in one of our laboratories were used to construct the libraries (49). Briefly, RNA was isolated using TRIzol and size fractionated using polyethylene glycol (PEG) precipitation. The low-molecular-weight fraction was electrophoresed ona 15% polyacrylamide–8 M urea gel, and small RNAs (20 to 27 nt) were extracted from the gel and purified. Both 5 and 3 RNA adapters (Dharmacon Research, Boulder, CO) were sequentially ligated onto the RNA using T4 RNA ligase (Ambion, Austin, TX). The 5 RNA adapter (5-OH-GGUCUUAGUCG CAUCCUGUAGAUGGAUC-OH 3) and 3 RNA adapter (5-P-CACUGAU GCUGACACCUGC-idT-3; idT is inverted deoxythymidine) were designed to prevent self-ligation, and the ligation products were purified following each step. The RNA was then reverse transcribed (Superscript reverse transcriptase [RT]; Invitrogen) using a primer complementary to the 3 adapter. cDNA inserts were amplified by PCR using primers corresponding to both adapters. Amplicons were sequenced by using the 454 Life Sciences system that utilizes pyrosequencingbased, sequence-by-synthesis, high-throughput, parallel sequencing (52). Sequence data were filtered for adapter sequences and clustered (allowing a 4-base overhang or mismatch at either end), and the insert sequence was analyzed by comparing it to the chicken and MDV genomes and to the miRNA database (55) using BLAST (3). |
回复此楼» 猜你喜欢
一个有机合成实验室都需要哪些设备？
已经有6人回复
-
2026年国自然面上资助率
已经有20人回复
-
面上再次挂了，太难了，躺也躺不了，倦也卷不过，小学校之殇！
已经有22人回复
-
微信指数没变化，科研之友没阅读
已经有18人回复
-
基础研究怎么拉横向，学校到款任务越来越多，难以完成 拉横向，都有哪些途径啊
已经有8人回复
-
HXDI做水性聚氨酯乳液，是不是特别容易出渣
已经有3人回复
-
系统今天又提示维护了，估计离放榜不远了
已经有15人回复
-
你们的时间戳变了吗
已经有4人回复
-
产物和副产物价值比较
已经有5人回复
-
时间戳他又来了
已经有17人回复
2楼2010-04-27 20:33:39
3楼2010-04-28 09:15:02
4楼2010-04-28 09:23:09
5楼2010-05-08 10:46:45