User:Alexander S Mikheyev/Notebook/rotifer alien genes/2009/11/19

Alien genes

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To do Oil flies prepare for sequencing Order: GITC Submit AmNat paper PEG precipitation recipe: 3g PEG 8000 2.19g NaCl Bring up to 15ml with good water. added 8ul PEG to PCR spun at max speed for 15 min removed PEG, added 150ul cold 80% ethanol spin 7 min remove ethanol, dry re-suspend in 10 ul water nanodrop Sample Nucleic Acid Conc. Unit 260/280 260/230 intergenic 12 ng/µl 1.95 0.62 AV10007 11.2 ng/µl 1.85 0.67 AV10016 13.6 ng/µl 1.81 0.67 AV10027 10.2 ng/µl 1.95 0.88 AV10153 8 ng/µl 2.34 1.53 PR10008 13.8 ng/µl 1.69 2.18 somehow I don't feel completely comfortable with potential effects of CoraLoad dye on precipitation, so I'll run a gel: InstaGene matrix for extraction of small numbers of rotifers placed 1, 2, 4 and 8 A. ricciae into 30 ul instagene buffer 30 min@ 56, with one vortex, 8 min at 99C Reagents 1x conc. 9 h2o 4.75 - 42.75 10x 1 10x 9 dye 1 10x 9 dNTP 0.2 10 mM 1.8 Intergenic F 1 10 uM ea 9 Taq 0.05 5 U/um 0.45 DNA 2 20 ng/ul TOTAL 10 Cocktail per vial 8 1ul and 2 ul extraction 1 rotifer 2 rotifers 4 rotifers 8 rotifers 3m 94C (10s 94C; 3m 60C)x35 try one set with 1 ul DNA, and another set with 2 ul DNA 3ul PCR product 2ul ladder 1ul 4 rotifer lane -- no sample loaded into PCR