User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/03/25

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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 200ul 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 fluorescent microscope.

RESULTS Visualized on March 31. Still Alive and Stained.

POST-PCR Clean Up with Ethanol

Test on March 4, 2010 AV PCR product.

Procedure

(1) For each 10 ul of PCR product, prepare the following mixture in strip-cap tube.

  1 ul of 3M sodium acetate (NaOAc) pH 5.2
  25 ul chilled absolute ethanol. 

(2) Transfer the PCR product (20ul) into the tube containing the mixture. (3) Vortex the tube and then store it at -20 for 30-50 min. (4) Spin for 30 min at maximum speed 14.000 rpm. (5) Rremove the supernatant carefully and don't disturb the pellet. (6) Wash the pellet with 70% ethanol. And spin the tube at maximum speed for 5 min. (7) Remove the supernatant and then dry the pellet – air dry. (8) Resuspend the pellet in 10 ul in sterile water.


Original PCR : loaded 2.0ul of each sample Ethanol Cleaned : loaded 1.0ul of each sample

04March10 Original PCR.jpg 25March10 Ethanol.jpg

Alien Genes New Primer Screen

Reaction mixture conc. 1x x8.5 x2 x10.5 x2 ' upper ' '
Temp ?? 2 17 No. Sample Primer mix
TopTaq Buffer 10x 1 8.5 10.5 1 Tsukuba 3 D2 AV10134
dNTP mix 10 mM each 0.2 1.7 2.1 2 AV10121
Primer mix 5 uM each 1 10.5 3 AV10084
TopTaq 5 U/ul 0.05 0.425 0.525 4 AV10045
Milli Q 5.75 48.875 60.375 5 AV10044
6 AV10034
PCR cycle (2 STEP) TaKaRa Dice 7 AV10027
94C 3 min 8 AV10025
9 Tsukuba 3 D3 AV10134
98C 10 sec 10 AV10121
60C 1 min 11 AV10084
x35 12 AV10045
13 AV10044
72C 5 min 14 AV10034
15 AV10027
16 AV10025
lower
No. Sample
1 Tsukuba 3 D4 AV10134
2 Woods Hole 1 A6
3 Woods Hole 1 C2
4 Woods Hole 1 C3
5 Yokohama B5
6 Baltimore A3
7 Baltimore A6
8 E. Windsor D4
9 Harvard A1
10 Ithaca 2 A6
11 Tsukuba 3 D4 AV10025
12 Woods Hole 1 A6
13 Woods Hole 1 C2
14 Woods Hole 1 C3
15 Yokohama B5
16 Baltimore A3
17 Baltimore A6
18 E. Windsor D4
19 Harvard A1
20 Ithaca 2 A6


loaded 2.0ul of each sample

0046-1.jpg


Reaction mixture conc. 1x x97 ' No. Primer mix
Temp (49 - 60) ?? 2 194 1 AV10134
TopTaq Buffer 10x 1 97 2 AV10121
dNTP mix 10 mM each 0.2 19.4 3 AV10084
Primer mix 5 uM each 1 4 AV10045
TopTaq 5 U/ul 0.05 4.85 5 AV10044
Milli Q 5.75 557.75 6 AV10034
10 ul 7 AV10027
8 AV10025
PCR cycle
94C 3 min No.
61 Pennsylvania (PA) B3
98C 10 sec 62 Ithaca 1 C3
60C 3 min 63 Ithaca 1 C6
x35 64 Harvard A4
65 Ithaca 1 B4
72C 5 min 66 Ithaca 1 C5
67 Baltimore B2
68 Baltimore B3
69 Baltimore B6
70 E. Windsor C3
71 Pennsylvania (PA) D6
72 Baltimore B5


loaded 2.0ul of each sample

0049.jpg