L4440线虫干扰质粒

• 保存条件：负20度
• 保质期：2年
• 供应商：华纳生物
• 规格：2ug

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 adult 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
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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