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

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Alien Gene LongPCR 50ul RXN

' [CONC] 1X 15
Water 5.3 79.5
Takara LA Taq 5U 0.1 1.5
Takara LA Buffer 10X 1 3
dNTP 2.5uM each 1.6 24
*Template 1ng 1 15
Primer Mix 5uM each 1
10.0ul 135
add 1.0 Primer Mix
* AV or CCA3
PCR Cycle
94C 1 min
98C 10 sec
65C 15 min
30X
AMP Lane Template Contents ' Fosmid MIX
1 CCA3 AV10034 FWD AV10039 RVS AV240_B 40
2 CCA3 AV10039 FWD AV10041 FWD AV240_B 50
3 CCA3 AV10106 FWD AV 10109 FWD AV_TEL_L_B 54

Load 2.0ul sample File: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.