User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/04/09

Alien genes

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Individual Rotifer Extraction fresh from Moss sample 1) Soak fresh Moss sample in petri dish with autoclaved/filtered spring water - enough to cover sample. 2) Wait 1 hour and/or move sample around in water. 3) Search for rotifers. Maybe helpful to remove moss to search. 4) Onto a petri-dish, place 1 rotifer in 50ul of autoclaved/filtered spring water. Rinse 3 to 5 times with fresh autoclaved/filtered spring water, taking care not to suck up rotifer. Attempt to remove all debris leaving only single rotifer. 5) Pick rotifer with ~50ul of clean autoclaved/filtered spring water and place in extraction tube. 6) Proceed with Instagene extraction. Reaction mixture conc. 1x 10 ' ' AMP Lane Template Template Instagene ? 1 1 IRP 1 TopTaq buffer 10x 1 10 2 IRP 2 dNTP mix 10 mM each 0.2 2 3 IRP 3 Primer mix 5 uM each 0.5 5 4 IRP 4 TopTaq (QIAGEN) 5U/ul 0.05 0.5 5 IRP 5 Milli Q water 7.25 72.5 6 IRP 6 10 ul 90 9.0ul / RXN 7 Postitive Control CCA3 *Control AV 1ng/ul 8 Negative Control PCR cycle 94C 3 min 94C 10 sec 45-68C 3 min RAMP 0.5C/sec x35 68C 5 min Loaded 2.5ul PCR product LCO/HCO Reaction mixture conc. 1x 19 ' ' AMP Lane Template ' Template Instagene ? 1 A Boston A1 TopTaq buffer 10x 1 19 B Boston A4 dNTP mix 10 mM each 0.2 3.8 C Cape Cod (CC) A1 Primer mix 5 uM each 1 19 D Woods Hole 3 A5 TopTaq (QIAGEN) 5U/ul 0.05 0.95 I Woods Hole 3 C2 Milli Q water 6.75 128.25 75 Baltimore A5 10 ul 171 9.0ul / RXN 76 Pennsylvania (PA) B1 *Control AV 1ng/ul + Positive Control AV - Negative Control PCR cycle 94C 3 min 94C 10 sec 45-68C 3 min RAMP 0.5C/sec x35 68C 5 min Loaded 2.5ul PCR product Alien Genes New Primer Screen Reaction mixture conc. 1x x16.5 ' lane ' Temp ?? 2 27 Ithaca 2 A3 TopTaq Buffer 10x 1 16.5 28 Ithaca 2 A4 dNTP mix 10 mM each 0.2 3.3 29 Ithaca 2 A5 Primer mix (HIS F/R1) 5 uM each 1 16.5 37 Tsukuba 3 D2 TopTaq 5 U/ul 0.05 0.825 40 Woods Hole 1 A6 Milli Q 5.75 94.875 43 Yokohama B5 10 ul 47 Harvard A1 48 Ithaca 2 A6 PCR cycle 50 Tsukuba 1 B5 94C 3 min 51 Yokohama A2 55 Yokohama B2 98C 10 sec 56 Yokohama A4 60C 3 min 61 Pennsylvania(PA) B3 x35 62 Ithaca1 C3 75 Baltimore A5 72C 5 min 76 Pennsylvania (PA) B1