DNA extraction for PacBio
Attempt 3 to get genomic DNA for PacBio sequencing from Aphaenogaster.
Modified the ant phenol-chloroform DNA extraction protocol combined with the Thermo Scientific Phenol/Chloroform Extraction and Ethanol Precipitation protocol.
- Made STE buffer (0.1 M NaCl / 0.05 M Tris pH8 / 1mM EDTA)
- for 100ml:
- 10 ml 1M NaCl
- 0.2 ml 0.5M EDTA
- 84.8 ml H2O
- Collected about 50 ants (weight = 0.27 g) from Molly Bog colony JSG002
- Flash froze in liquid nitrogen
- Crushed ants with mortar and pestle (bleach sterilized)
- Mixed sample with STE + 0.5% SDS + 200ug/ml Proteinase K -for 10 ml:
- 9.42 ml STE
- 0.5 ml SDS 10%
- 82.98 ul Proteinase K (24.1 mg/ml)
- NOTE: the 10 ml digestino volume was way too much. I used this erroneously based on ‘DNA extraction for spermathecae’ protocol which listed ~3.8ml for 50 eggs. This measure was actually the volume to make, using only 50 ul per egg. I probably could have used only 1-2 ml of the mixture.
- Incubated overnight at 55°C
Example of how to reproduce old R scripts: [http://blog.revolutionanalytics.com/2014/08/gran-and-switchr-cant-send-you-back-in-time-but-they-can-send-r-sort-of.html]
Are these better than packrat
- packrat is for the future…stores packages at the time of your work
- GRAN is more multi-purpose…allows you to roll-back to any previous version of R packages