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产品名称：L4440
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简单介绍

L4440菌株发货前均经过严格的多重验证，如存在质量问题，请在收到产品的三个月内通知我司。L4440收到质粒后请短暂离心，加入20μl ddH2O溶解质粒，取2μl转化至对应感受态中，挑取单克隆重新提取质粒后使用。

产品描述

L4440

产品信息
基本信息
质粒简介
质粒图谱
质粒序列

产品信息

产品货号	产品名称	产品规格	优惠价
XY0311	L4440	20μl	¥请询价

使用说明

信裕质粒平台的各批次质粒菌株发货前均经过严格的多重验证，如存在质量问题，请在收到产品的三个月内通知我司。收到质粒后请短暂离心，加入20μl ddH2O溶解质粒，取2μl转化至对应感受态中，挑取单克隆重新提取质粒后使用。如需获取其他详细信息请登录我司官网查询。

基本信息

启动子:	T7
复制子:	pUC ori，F1 ori
质粒分类:	线虫RNAI干扰载体；Worm Expression RNAi
质粒大小:	2790bp
质粒标签:	lacZN, OriF1
原核抗性:	氨苄青霉素Ampicillin
克隆菌株:	DH5α
培养条件:	37℃，有氧，LB
表达宿主:	线虫，worm
诱导方式:	IPTG
5'测序引物:	根据全序列设计

质粒简介

Reverse Genetics and RNAi：
RNAi is a natural cellular process in many eukaryotes which scientists have taken advantage of in the lab as a valuable reverse genetics mechanism for regulating gene expression. RNAi involves double-stranded RNA (dsRNA) interfering with the expression of genes with sequences complementary to the dsRNA. By administering a specific dsRNA to a cell culture or multicellular organism, RNAi can be induced to selectively reduce expression of that specific gene in the cells or organism.
RNAi has been particularly well studied in C. elegans, in which the RNAi gene silencing phenotype is heritable and the dsRNA is easily administered. E. coli bacteria carrying RNAi plasmids that produce the desired dsRNA can be fed to worms, and the dsRNA is transferred to the worm via the intestinal tract. RNAi plasmids typically consist of DNA coding sequence from the intended target gene cloned between two T7lac promoters. The plasmid also has a selectable marker that confers resistance to an antibiotic, in this case ampicillin. In 7.15, you will use the E. coli strain HT115 carrying various L4440 plasmids, each containing a different cloned gene sequence. HT115 is an RNase III-deficient E. coli strain with IPTG-inducible T7 polymerase activity. To induce dsRNA production from these plasmids, the HT115 bacteria is grown on special RNAi NGM feeding plates that contain IPTG and the ampicillin analog carbenicillin. Carbenicillin is preferred over ampicillin because it tends to be more stable.
Protocol
A. Preparing feeding plates
1) Inocluate 3mL of LB containing 50 µg/mL ampicillin with individual desired bacterial strain. Grow overnight at 37^oC.
2) Seed NGM agar feeding plates (containing 25 µg/mL carbenicillin and 1mM IPTG) by pipetting 150 µL of LB bacterial culture into the center of the plate. You should have three RNAi feeding plates:
a. One plate will be seeded with HT115 bacteria carrying the “empty” L4440 vector (no C. elegans gene is cloned in the vector).
b. Another plate will be seeded with HT115 bacteria carrying a portion of the unc-22 gene cloned into the L4440 vector. The phenotype resulting from RNAi of unc-22 is well-established and is reliably reproducible under the experimental conditions you are using today.
c. A third plate will be seeded with HT115 bacteria carrying a portion of your gene of interest cloned into the L4440 vector. We will use dpy10 as our gene of interest.
3) Let the plates dry overnight at room temperature.
B. Transfer N2 L4 worms
1) Transfer two L4 hermaphrodites from the N2 stock plate to each of the RNAi feeding plates, minimizing the amount of OP50 bacteria transferred. Try to avoid bringing any younger contaminating worms along with the L4 worms you are transferring.
2) Incubate the plates until the progeny reach the desired age (Note: it takes ~4 days for a C. elegans egg to grow into a gravid ***** when grown at 16^oC.
C. Scoring RNAi phenotypes
1) Observe RNAi controls. Start by looking at the L4440 RNAi plates - what phenotype(s) would you expect to see on these plates? Next, look at the unc-22 RNAi plates – what phenotype(s) would you expect to see on these plates? Record all your observations in your notebook. If you do not see the expected phenotypes on your control plates, this may indicate your RNAi experiment was set-up incorrectly.
2) Observe the RNAi phenotypes for the experimental RNAi construct and record all your observations in your notebook. Examine how the observed phenotypes compare with the corresponding null mutant phenotypes in the same gene (the WormBase database has information on previously characterized RNAi and null mutant phenotypes).

质粒图谱

质粒序列

LOCUS Exported 2790 bp ds-DNA circular SYN 04-SEP-2016

DEFINITION synthetic circular DNA

ACCESSION .

VERSION .

KEYWORDS L4440

SOURCE synthetic DNA construct

ORGANISM synthetic DNA construct

REFERENCE 1 (bases 1 to 2790)

AUTHORS .

TITLE Direct Submission

JOURNAL Exported Sunday, September 4, 2016 from SnapGene Viewer 3.1.4

http://www.miaolingbio.com

FEATURES Location/Qualifiers

source 1..2790

/organism="synthetic DNA construct"

/mol_type="other DNA"

promoter 19..37

/note="T7 promoter"

/note="promoter for bacteriophage T7 RNA polymerase"

primer_bind 95..111

/note="SK primer"

/note="common sequencing primer, one of multiple similar

variants"

primer_bind complement(181..197)

/note="KS primer"

/note="common sequencing primer, one of multiple similar

variants"

promoter complement(223..241)

/note="T7 promoter"

/note="promoter for bacteriophage T7 RNA polymerase"

primer_bind complement(251..267)

/note="M13 fwd"

/note="common sequencing primer, one of multiple similar

variants"

rep_origin 409..864

/direction=RIGHT

/note="f1 ori"

/note="f1 bacteriophage origin of replication; arrow

indicates direction of (+) strand synthesis"

promoter 890..994

/gene="bla"

/note="AmpR promoter"

CDS 995..1855

/codon_start=1

/gene="bla"

/product="beta-lactamase"

/note="AmpR"

/note="confers resistance to ampicillin, carbenicillin, and

related antibiotics"

/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI

ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS

PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW

EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA

LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS

LIKHW"

rep_origin 2026..2614

/direction=RIGHT

/note="pUC ori"

/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of

replication"

ORIGIN

1 aacctggctt atcgaaatta atacgactca ctatagggag accggcagat ctgatatcat

61 cgatgaattc gagctccacc gcggtggcgg ccgctctaga actagtggat ccaccggttc

121 catggctagc cacgtgacgc gtggatcccc cgggctgcag gaattcgata tcaagcttat

181 cgataccgtc gacctcgagg gggggcccgg tacccaattc gccctatagt gagtcgtatt

241 acgcgcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc

301 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc

361 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgggac gcgccctgta

421 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca

481 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct

541 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc

601 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat

661 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc

721 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc

781 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta

841 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc

901 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg

961 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc

1021 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt

1081 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct

1141 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac

1201 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact

1261 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa

1321 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga

1381 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt

1441 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga

1501 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg

1561 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat

1621 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat

1681 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc

1741 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga

1801 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc

1861 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag

1921 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc

1981 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt

2041 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt

2101 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat

2161 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc

2221 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa

2281 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg

2341 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag

2401 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag

2461 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa

2521 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt

2581 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg

2641 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc

2701 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac

2761 cgagcgcagc gagtcagtga gcgaggaagc

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产品目录

产品资料

L4440

L4440

产品信息

使用说明

基本信息

质粒简介

质粒图谱

质粒序列

产品目录

产品资料

L4440

L4440

产品信息

使用说明

基本信息

质粒简介

质粒图谱

质粒序列

相关产品