title: MITOMI 2.0 JokerCake 8mer library (microfluidic affinity assay)
status: Public on Jun 19 2017
contact.city: Stanford
contact.first: Polly
contact.middle: Morrell
contact.last: Fordyce
contact.email: pfordyce@stanford.edu
contact.state: CA
contact.address: 443 Via Ortega
contact.department: Genetics, Bioengineering
contact.country: USA
contact.institute: Stanford University
contact.phone: 6507998131
organism: synthetic construct
description: de Bruijn Joker synthetic library
  spotted oligonucleotide with aligned microfluidic device
  MITOMI 2.0 experiment, 240 oligonucleotides, 4 replicates per oligonucleotide
  The MITOMI 2.0 JokerCake 8mer library is a library of synthetically designed sequences designed to include all possible 8mer DNA sequences in a minimally compact space by including degenerate 'joker' characters (N = approx. equimolar mixture of A, C, G, and T). These designed library sequences are ordered as linear oligonucleotides from IDT and then converted to Alexa-647-labeled dsDNA via annealing of an Alexa-647-labeled universal probe to the 3' end of each oligonucleotide, followed by extension using Klenow Fragment, exonuclease -.
manufacture_protocol: 1. Oligos are ordered from IDT in shalllow 96 well plates.  Each well is concentration- and volume-normalized to contain 50 uL of 100 uM oligonucleotide.
  2. Create a daughter plate at 25 uM by removing 5 uL of 100 uM oligonucleotide and adding 15 uL ddH20.
  3. Convert DNA to fluorescently-labeled dsDNA via steps 4-7.
  4. In each well of a new 96 well plate, combine 1.5 uL dNTPs at 10 mM each, 1.5 uL ddH20, 1.0 uL NEBuffer 2, 3.0 uL library primer at 25 uM, and 3.0 uL 25 uM Alexa-647-labeled primer.
  5. Melt DNA at 95C for 3 minutes, slow anneal to 25C over 45 minutes.
  6. To each well, add 0.2 uL Klenow exo-, 0.5 uL NEBuffer 2, and 4.3 uL ddH20.
  7. Heat to 37C for 60 minutes for Klenow extension; deactivate Klenow by heating to 72C for 20 minutes.
  8. Transfer fluorescently-labeled dsDNA to a 384 well plate and dilute to 1.25 uM DNA in a solution of 2% BSA and 12.5 mg/mL trehalose in 3x SSC.
  9. Print fluorescently-labeled dsDNA onto ThermoFisher SuperChip epoxysilane-coated slides.
  10. Follow standard MITOMI device protocols for surface treatments and measurements.