Etchevers:Notebook/STRA6 in eye development/2008/04/17

Genetics of human eye development

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DNA extraction from mouse tails Angelique is trying to PCR amplify a fragment of mouse genomic DNA that will serve as a template for synthesizing a new RNA probe for in situ hybridization against Pax6. She kept getting very bad yields. We looked with the spectrophotometer at the genomic DNA concentration and the dilution was way low. We then looked at the stock solution and it was 20x less than indicated. She tried putting all the dilute DNA instead of the water in the next PCR reaction but still a bad yield. Meantime, I had some frozen pups and was able to break off a bit of tail to extract the DNA. Put tissue in 2ml Eppie tube with: 240 μL 10% SDS 100 μL 20 mg/ml Proteinase K 200 μL 10x PBS buffer 1.46 mL water Incubated at 50°C for 8 hours. This evening, all was dissolved except for (presumably) ossifying bits of the tail. What a great principle for a horror novel! Blech. Spun down 1 min at 10K rpm on benchtop centrifuge. Removed supernagent to 15 mL tube. Added 2 mL φ/CHCl3/IAA and mixed thoroughly. Spun in large centri 2 minutes at 3500 rpm. Supernagent to new 15 mL tube. Adde 2 mL CHCl3. Mixed. Spun in large centri 2 minutes at 3500 rpm. Decant supernatant to new tube. Added: 25 μL 3M Na acetate 5 mL ice-cold 100% EtOH Agitated and got lovely threaded DNA precipitate. Fished it out with loop into 2 mL tube again. Rinsed with 1 mL ice-cold 70% EtOH. Let dry the pellet about 15 min at RT. Added 1 ml Tris-EDTA (10 mM/ 1 mM respectively). Agitate overnight to aid in resuspension. Drove Kyoko all over town to look for an apartment for her. Only incidentally related to eye project in that she's here to do linkage studies for the high myopia project. Alethea 13:44, 17 April 2008 (EDT):