User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/03/18

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Alien Gene LongPCR 50ul RXN

' [CONC] 1X 15
Takara LA Taq5U0.11.5
Takara LA Buffer10X13
dNTP2.5uM each1.624
Primer Mix5uM each1
add 1.0 Primer Mix
* AV or CCA3
PCR Cycle
94C 1 min
98C10 sec
65C15 min
AMP Lane Template Contents ' Fosmid MIX
1CCA3AV10034 FWDAV10039 RVSAV240_B40
2CCA3AV10039 FWDAV10041 FWDAV240_B50
3CCA3AV10106 FWDAV 10109 FWDAV_TEL_L_B54

Load 2.0ul sample Image:  18mar10.tif

SNARF-1 and RED Rotifer Staining

- A. vaga - ~10-20 rotifers per treament

TREATMENT Test 200ul of 1) 1:800 dye dilution

  a)1 hour

Stock tubes at 50ug - diluted with 9ul of DMSO then diluted to 1:800 with MiliQ pure water

(1) Transfer 3 ml of spring water (pH 4.5-5) to a glass vessel (small glass petri dish or culture tube).

(2) Add 200 µl of 10 mM dye stock. Mix thoroughly. (To make 10 dye stock, resuspend an aliquot containing 50 µg CFDA-SE in 9 µl ultrapure DMSO. Store frozen at –20oC.)) - saved used dye for restaining.

(3) Add healthy, well fed animals to be stained in 40ul total volume (a 1:800 final dilution of dye; 25 µM final concentration)

(4) Incubate at room temperature for 1 hour in a dark location.

(5) Pipette off media in the staining dish and add 200ul filtered spring water. Repeat rinse 3-5 times. Rinsing in the dish with retain most of the adults while eggs and juveniles which do not attach as well may be lost. To retain younger animals or if staining in a glass tube: transfer stained animals to a centrifuge tube; spin to pellet all animals; discard supernatant, taking care to not disrupt the animal pellet; add filtered spring water. Repeat 3-5 times.

(6) Incubate the stained animals for 15 minutes at room temperature in 200ul filtered spring water.

(7) Visualize on a fluorescent microscope.

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