mRNA Library Prep: cDNA clean up through adapter ligation
Deoxynucleotide Mix T4 DNA polymerase and Klenow enzyme converts overhangs into blunt ends T4 DNA Polymerase T4 is a bacteriophage of E. coli. E. coli DNA Polymerase I, 3 ʹ to 5 ʹ exonuclease activity of Large (Klenow) Fragment enzymes removes 3 ʹ overhangs and polymerase activity fills in 5 ʹ overhangs. T4 Polynucleotide Kinase T4 PNK phosphorylates 5 ʹ ends of DNA fragments Reagents Their Role in the Reaction
Deoxyadenosine 5 ́ - Prevents fragments from ligating to one another during Triphosphate (1 mM dATP) adapter ligation reaction Klenow Fragment (3 ́→ 5 ́ Corresponding single ‘T’ exo–) nucleotide on 3’ end of adapter provides a complementary overhang for ligating adapter to fragment To ensure a low rate of chimera (concatenated template) formation Reagents Their Role in the Reaction
DNA Adaptors (15 µM) Adds distinct sequences to the 5’ and 3’ ends of each strand in the genomic fragment Quick T4 DNA Ligase Additional sequences added by tailed primers during PCR Additional sequences are necessary for library amplification on flow cell during cluster formation Reagents Their Role in the Reaction
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