Friday, December 20, 2019

Csa Concentration Varied Between Rounds ( Table 1 )

CsA concentration varied between rounds (Table 1). Library was ordered from Integrated DNA Technologies (IDT). N30 library (30 unknown nucleotide bases): 5 -GGA GGC TCT CGG GAC GAC NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN GTC GTC CCG ATG CTG CAA TCG TAA-3 . Capture Sequence: GTC GTC CCG AGA GCC ATA/3BioTEG/. Primer_P1: GGAGGCTCTCGGGACGAC. Primer_P2: TTACGATTGCAGCATCGGGACG. P2_biotin: /5BiosG/TTACGATTGCAGCATCGGGACG. All oligo’s (primers, library, and capture-strand) were ordered from IDT . Bio-capture used: 5’ biotin from IDT. Two SELEX buffers were used: SELEX 1 buffer (10 mM HEPES, 150mM NaCl, 5mM KCl, and 2mM MgCl2) and SELEX 2 buffer (10mM HEPES, 150mM NaCl, 5mM KCl). The composition of the PCR mix was as follows: 10% 10X buffer, .5% Taq, 1% P1, 1% P2, 2% dTNP, and 80.5% water. All gels were run in .5X TBE buffer and 3% agarose gel. Columns used: Micro Bio-Spinâ„ ¢ Chromatography Columns. PCR polymerase used was Choice Taq from Denville Scientific. DNTP is also fro m Denville Scientific. Streptavidin Beads are from Sigma Aldrich. 4.2 The SELEX Process The general steps of the SELEX process are; the binding reaction between the aptamers and the target, washing steps to remove the unbound DNA, PCR amplification of the bound DNA, and strand separation of the DNA back into ssDNA [3]. The Capture-SELEX cycle consists of three main sections: Elution profile, Library Amplification, and Strand Separation. The elution profile section includes the adding of biotin to the library

No comments:

Post a Comment

Note: Only a member of this blog may post a comment.