Haynes:LCR Assembly

LCR Protocol Development

• Ref Cameron's last successful attempt: http://openwetware.org/wiki/Haynes_Lab:Notebook/Engineering_PC-TFs/2014/12/22
• Use this info to develop a protocol
• HOLD: need to order oligos to amplify MV10 (~30 nt, w/ 5' phosphates, high Tm)
  • Should not mix PCR-amp fragments with digested fragments

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