User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/06

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04/06/15

  • LCR - continued
  • Order new oligos - MV10 (5' phos)



LCR Protocol Development


Part 1: Prep the Oligo Bridges

  • Note: Final conc. in LCR rxn. is 30 nM each
  • Bring the IDT oligo pellet to 100μM with dH2O. nmoles oligo (on label) * 10 = μL H2O to add
  • Make a 300 nM working solution (final volume = 100 μL) in a new tube. 3 μL of 100μM oligo stock + 97 μL dH2O = 100 μL
  • Use 1.0 μL of oligo working sln. per 10 μL LCR reaction


Part 2: Prep the DNA fragments (PCR)

  • Note: Final conc. in LCR rxn is 3 nM each
  • Use primers with 5' phosphates to amplify the fragment(s) of interest in 50 μL Phusion polymerase PCR reactions. Use Phusion polymerase to generates fragments with blunt ends (others produce T/A overhangs).
  • OPTIONAL - Digest the template DNA: Add 1 μL FastDigest DpnI and 5μL of 10x FastDigest buffer to each PCR reaction. Incubate at 37°C for 15 min.
  • Purify the product with a kit of choice (e.g. Sigma PCR clean-up)
  • Measure the ng/μL of the purified sample.
  • Dilute the purified dsDNA to 30 fmol/μL (30 nM)
    • The volume of purified DNA (x) you will need to dilute in a final volume of 50 μL = length in bp ÷ measured ng/μL * 30 fmol/μL * 650 fg/fmol dsDNA ÷ 1,000,000 fg/ng * 50 μL final volume.
    • Formula: x μL = length in bp ÷ measured ng/μL * 0.0195 ng/μL * 50μL
  • Use 1.0 μL of diluted dsDNA per 10 μL LCR reaction


Notes:

  • Cameron used ~1μL of ~24 ng/μL insert (restriction digest)
  • This measured ng/μL is too low for the dsDNA dilution step (x = ~90, which is greater than 50)
  • Cameron used ~36.93 fmol in a 25 μL LCR = 1.47 fmol/μL (1.47 nM), whereas the recommended amount is 3.0 fmol/μL (3 nM)
  • Also appears that Tm's were not optimized to 60°C for each half of the bridge oligo



New Oligos

  • Add 5'-phos for LCR
  1. MV10 r1, 5'phos-ctccatggtggcggcgggactagactcgag
  2. MV10 f1, 5'phos-cccaagaaaaagcgcaaggtacaccatcac



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