Jeff:LabNotes/Microbiome/2011-10-24
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Colony Prep[edit]
- Purify with minelute
Ligation
- I selected for 300-600 bp, so the mean size is ~450 bp. This corresponds to 32 ng DNA/rxn
- Create the following: 32 ng DNA, 1 uL buffer, 1 uL vector, 1 uL t4 ligase, fill to 10 uL with water
- Incubate at 16 C for 1 hr
Transfection
- Add 1 uL of ligated product to thawed competent cells
- Incubate on ice for 30 min
- Heat shock at 42 C for 30 s
- Put back on ice for 2 min
- Add 250 uL SOC media to each and incubate at 37 C for 1 hr with shaking
- Add 20 uL XGAL to each plate (warm up plates and XGAL to rt) and incubate in dark for 10 min
- Add 50 uL cells to each plate and incubate overnight at 37 C