Janet B. Matsen:Lab Tips & Tricks
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I came to the Lidstrom Lab with zero molecular biology experience. I have great appreciation for all of the resources put online by the OpenWetWare community and other websites, so I am hopeful that I can share some of what I have learned. Please contact me with questions, comments, and corrections.
This collection is largely advice given to me by The Amazing Justin Siegel.
- purifying DNA on a gel column has ~ 90% loss. purifying DNA on a column (non-gel) has ~ 90% recovery. avoid gel purification. (Justin)
- Freeze 'N Squeeze Gel extraction...?
- Purify gels on gel-specific kits. Purify PCR products on PCR product specific kits. The former does a less effective job removing primer dimers. (Justin)
- Try annealing = 55oC in PCR before anything else.
- Grow in TB, not LB. This along gives me orders of magnitude higher miniprep yields.
- Grow overnight cultures in liquid:air ratios of at least 1:4; increasing the culture volume seems like a good idea at first but the lower oxygen concentrations are detrimental to plasmid yields.
- LB gives you more cells than TB
- Use TB (1-2 mL) overnight for plasmid preps (for pSB1A3, pSB3K3)
- Don't transform plates at room temp over the weekend, especially if you rely on Amp resistance. Often you see a lot of contamination! (Janet)
- Don't be surprised if you get contamination on plates with multiple antibiotic resistances. There are plenty of plasmids floating around natural systems with multiple on them already. Bacteria make antibiotics in natural systems to add competition to an an environment. (Mary Lidstrom)