Improved cloning efficiency of polymerase chain reaction (PCR) products after proteinase K digestion

Crowe, J.S.; Cooper, Helen J.; Smith, M.A.; Sims, Martin; Parker, D; Gewert, Dirk R.

doi:10.1093/nar/19.1.184

Cited by 133 publications

(37 citation statements)

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“…In order to generate discrete fragments of the larger E. gracilis rRNAs we adapted an RNase protection approach (Casey and Davidson 1977;Van Stolk and Noller 1984), using PCR products spanning the regions of interest. Taq polymerase was removed from the PCR products by digestion with proteinase K, phenol extraction, and ethanol precipitation (Crowe et al 1991;Barnes 1992).…”

Section: Modified Nucleotide Mappingmentioning

confidence: 99%

Pseudouridine-guide RNAs and other Cbf5p-associated RNAs in Euglena gracilis

Russell¹,

Schnare²,

Gray³

2004

RNA

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In eukaryotes, box H/ACA small nucleolar RNAs (snoRNAs) guide sites of pseudouridine (⌿) formation in rRNA. These snoRNAs reside in RNP complexes containing the putative ⌿ synthase, Cbf5p. In this study we have identified Cbf5p-associated RNAs in Euglena gracilis, an early diverging eukaryote, by immunoprecipitating Cbf5p-containing complexes from cellular extracts. We characterized one box H/ACA-like RNA which, however, does not appear to guide ⌿ formation in rRNA. We also identified four single ⌿-guide box AGA RNAs. We determined target sites for these putative ⌿-guide RNAs and confirmed that the predicted ⌿ modifications do, in fact, occur at these positions in Euglena rRNA. The Cbf5p-associated snoRNAs appear to be encoded by multicopy genes, some of which are clustered in the genome together with methylation-guide snoRNA genes. These modification-guide snoRNAs and snoRNA genes are the first ones to be reported in euglenid protists, the evolutionary sister group to the kinetoplastid protozoa. Unexpectedly, we also found and have partially characterized a selenocysteine tRNA homolog in the anti-Cbf5p-immunoprecipitated sample.

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Section: Modified Nucleotide Mappingmentioning

confidence: 99%

Pseudouridine-guide RNAs and other Cbf5p-associated RNAs in Euglena gracilis

Russell¹,

Schnare²,

Gray³

2004

RNA

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“…The reaction mixtures were overlaid with 100 p1 of mineral oil and amplified for 30 cycles, each cycle consisting of a I-min denaturation period at 94"C, a 1-min anneal at 55"C, and a 1-min extension period at 72°C. The PCR product was treated with SDS and proteinase K prior to digestion and ligation [29]. The product was digested with BumHI and HindIII and ligated with digested plasmid pQE30 (Qiagen Inc.) to form pCF33, which encodes a fusion protein containing the truncated ClfA fused with an N-terminal segment containing six histidine residues.…”

Section: S Aureus Strains Strain Newman Expresses a High Level Ofmentioning

confidence: 99%

Characterization of the Interaction Between the Staphylococcus Aureus Clumping Factor (ClfA) and Fibrinogen

McDevitt¹,

Nanavaty²,

House-Pompeo³

et al. 1997

European Journal of Biochemistry

200

168

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The ability of Staphylococcus aureus to adhere to adsorbed fibrinogen and fibrin is believed to be an important step in the initiation of bioinaterial and wound-associated infections. In this study, we show that the binding site in fibrinogen for the recently identified S. aureus fibrinogen-binding protein clumping factor (ClfA) is within the C-terminus of the fibrinogen y chain. S. aureus Newman cells expressing ClfA adhered to microtitre wells coated with recombinant fibrinogen purified from BHK cells, but did not adhere to wells coated with a purified recombinant fibrinogen variant where the 4 C-terminal residues of the y chain were replaced by 20 unrelated residues. In addition, a synthetic peptide corresponding to the 17 C-terminal amino acids of the fibrinogen y chain effectively inhibited adherence of ClfA-expressing cells to fibrinogen. In western ligand blots, a recombinant truncated ClfA protein called Clf33 (residues 221 -550) recognized intact recombinant fibrinogen y chains, but failed to recognize recombinant fibrinogen y chains where the 4 C-terminal amino acids were altered by deletion or substitution. Previous studies have shown that the C-terminal domain of fibrinogen y chains contains a binding site for the integrin . We now show that Clf33 inhibits ADPinduced, fibrinogen-dependent platelet aggregation in a concentration-dependent manner and inhibits adhesion of platelets to immobilized fibrinogen under fluid shear stress, indicating that the binding sites for the platelet integrin and the staphylococcal adhesin overlap. The interaction between Clf33 and fibrinogen was further characterized using the BIAcore biosensor. When soluble Clf33 was allowed to bind to immobilized fibrinogen, a Kd of 0.51 t-0.19 pM was experimentally determined using equilibrium binding data. It was also shown that the synthetic C-terminal y-chain peptide effectively inhibited this interaction.

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“…PCR was performed using a Perkin-Elmer DNA thermal cycler 480, using the manufacturer-supplied protocols. PCR products were treated with proteinase K (BRL) to remove any possible attached DNA polymerase (Crowe et al, 1991). The 3' overhang at the end of the PCR products was removed by polishing with a T4 DNA polymerase.…”

Section: Amplification Of Flbr Cdna Fragment By Pcrmentioning

confidence: 99%