LCR Plan
As of yet, we haven’t been able to get LCR to work in our hands. The following are some trouble-shooting steps that have been taken, should be taken, or parameters that could be manipulated to optimized the protocol (read: get it to actually work)-
What has been tried:
- Gel extracting linearized MV10 from gel after digesting to avoid having heavy background of circular plasmid in LCR
- Re-amplifying all parts
What should be tried:
- Go back to MV10 and insert blunt site (SmaI CCCGGG or PmeI GTTTAAAC) as done in Karmella’s notebook from 03/14/13, make sure there’s no frame shift when orf goes in after Kozaks
- Could use MV9 and insert CMV promoter into spe/not cut site with xba/not or eco/pst to maintain Biobrick suffix and prefix
Manipulable parameters:
- Incubation temperature and time of MBN (suggested: room temperature, less than 1u of enzyme, 15 minutes)
- Phosphorylation of parts (heat substrate/buffer mix to 70C for 10min, chill on ice before adding ATP/enzyme then incubate at 37C)
- Does the vector need to be phosphorylated or not? Should still be phosphorylated after blunting? Do we need to dephosphorylate it to prevent reannealing? This could inhibit proper LCR though, as 5’ phosphate is necessary.
- Annealing temp (should not drop below melting temperature of oligo bridges, annealing temp should equal lowest bridge Tm)
- Concentrations and ratios of fragments and bridges (suggested: 6.2nM fragment DNA, 3.1nM oligo bridges)
- DMSO and betaine addition and concentrations
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