- 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
- 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
- MV10 r1, 5'phos-ctccatggtggcggcgggactagactcgag
- MV10 f1, 5'phos-cccaagaaaaagcgcaaggtacaccatcac