Database error: Invalid SQL: update pwn_comment set cl=cl+1 where id='6177' and iffb='1'
MySQL Error: 1142 (UPDATE command denied to user 'qdm791091491'@'114.215.125.138' for table 'pwn_comment')
#0 dbbase_sql->halt(Invalid SQL: update pwn_comment set cl=cl+1 where id='6177' and iffb='1') called at [/data/home/qxu1885310369/htdocs/includes/db.inc.php:73] #1 dbbase_sql->query(update {P}_comment set cl=cl+1 where id='6177' and iffb='1') called at [/data/home/qxu1885310369/htdocs/comment/module/CommentContent.php:54] #2 CommentContent() called at [/data/home/qxu1885310369/htdocs/includes/common.inc.php:518] #3 printpage() called at [/data/home/qxu1885310369/htdocs/comment/html/index.php:13] 网友点评--保定市万色网络科技有限公司
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Rst Strand CDNA, 0.2 M Of The Indicated 5` And 3` Oligonucleotides, 1X Advantage
Rst strand cDNA, 0.2 M of PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27919709 the indicated 5` and 3` oligonucleotides, 1X Advantage 2 PCR Buffer, 1X dNTP Mix, and 1X Advantage 2 Polymerase Mix. These reactions were performed on a Perkin-Elmer 9600 Thermocycler using the following cycling conditions: 72 for 10 min, 95 for 20 s, followed by 3 cycles of 95 for 5 s and 68 for 8 min. Primer extended cDNAs were visualized on an ethidium bromide stained 1.25 agarose, 1X TBE gel for quality assessment. They appeared as homogeneous smears ranging from 100 bp to 5 kb. mRNA aliquots not used for cDNA synthesis and cloning were also subjected to PCR amplification using Taq Polymerase and visualized on the same gel. The absence of any visible product on the gel confirmed thatPage 19 of(page number not for citation purposes)BMC Genomics 2006, 7:http://www.biomedcentral.com/1471-2164/7/genomic DNA did not contaminate these mRNA populations. Following proteinase K digestion and phenol:chloroform extraction, the amplified cDNAs were digested with 10 Sfi I (20 U/ ) at 50 for 2 h and size fractionated using CHROMA SPIN-400 columns (Clontech). The first three to four fractions containing cDNAs Sodium dichloroacetate longer than 500 bp were pooled, ethanol precipitated, and concentrated in 4.0 l nuclease free water (Gibco, UltraPure). These cDNAs were directionally cloned into Sfi I digested TripIEx2 (Clontech), and packaged using Gigapack III Gold Packaging Extract (Stratagene) according to the protocol provided. Packaged recombinant phages were incubated with log phase E. coli XL1-Blue cells (Stratagene), plated and library titers determined. All three libraries were plated at 100 and 1000 pfu/plate. White plaques were isolated and recombinant phages eluted overnight in 100 l SM buffer (0.1 M NaCl, 0.01 M MgSO4.7H2O, 0.05 M Tris-HCl (pH 7.5), 0.01 (w/v) gelatin). The inserts were amplified via PCR using 5` and 3` vector specific primers; 5` LD Amplimer Primer, 5`CTCGGGAAGCGCGCCATTGTGTTGG-3` and 3` LD Amplimer Primer, 5`-ATACGACTCACTATAGGGCGAATTGGC-3` (Invitrogen). Amplification reactions contained 0.4 l eluted phage, 0.03 pmol of each primer, 1X Taq Polymerase Buffer (Invitrogen), 3 mM MgCl2, 1 mM of each dNTP, and 0.2 U Taq Polymerase (Invitrogen) in a total volume of 25 ml. Reactions were performed in 96-well plates on a Perkin-Elmer 9700 Thermocycler using the following cycling conditions; initial denaturation at 95 for 5 min, followed by 25 cycles of denaturation at 94 for 30 s and annealing/elongation at 70 for 2 min, and a final elongation step PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28559105 at 68 for 3 min. Seven samples were chosen randomly from each 96-well plate amplified, and 5 ml of the reaction was electrophoresed on an ethidium bromide stained 1 agarose, 1X TBE gel to confirm that the PCR was not contaminated and that no primer dimers could be visualized (data not shown). 704 PCR-amplified cDNA clone inserts were visualized on ethidium bromide stained 1 agarose, 1X TBE gels, and their insert sizes determined using the KODAK Digital Science 1D software (Scientific Imaging Systems; data not shown).cDNA clone sequencing and EST assembly cDNA clones were picked at random from the S, RB and IRB abdomen libraries and their 5` end sequences obtained through single-pass sequencing of the PCRamplified inserts using the ABI PRISM Big Dye Terminators 3.0 Cycle Sequencing kit (ABI). All sequencing reactions were performed in 384-well plates on a PerkinElmer 9700 Thermocycler using the following cycling conditions; initial denaturation at 94 for 4 min, fol-l.
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