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

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Contents

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

Image:04March10 Original PCR.jpg Image:25March10 Ethanol.jpg

Alien Genes New Primer Screen

Reaction mixture conc. 1x x8.5 x2 x10.5 x2 ' upper ' '
Temp??217No.SamplePrimer mix
TopTaq Buffer10x18.510.51Tsukuba 3 D2AV10134
dNTP mix10 mM each0.21.72.12AV10121
Primer mix5 uM each110.53AV10084
TopTaq5 U/ul0.050.4250.5254AV10045
Milli Q5.7548.87560.3755AV10044
6AV10034
PCR cycle (2 STEP)TaKaRa Dice7AV10027
94C3 min8AV10025
9Tsukuba 3 D3AV10134
98C10 sec10AV10121
60C1 min11AV10084
x3512AV10045
13AV10044
72C5 min14AV10034
15AV10027
16AV10025
lower
No.Sample
1Tsukuba 3 D4AV10134
2Woods Hole 1 A6
3Woods Hole 1 C2
4Woods Hole 1 C3
5Yokohama B5
6Baltimore A3
7Baltimore A6
8E. Windsor D4
9Harvard A1
10Ithaca 2 A6
11Tsukuba 3 D4AV10025
12Woods Hole 1 A6
13Woods Hole 1 C2
14Woods Hole 1 C3
15Yokohama B5
16Baltimore A3
17Baltimore A6
18E. Windsor D4
19Harvard A1
20Ithaca 2 A6


loaded 2.0ul of each sample

image:0046-1.jpg


Reaction mixture conc. 1x x97 ' No. Primer mix
Temp (49 - 60)??21941AV10134
TopTaq Buffer10x1972AV10121
dNTP mix10 mM each0.219.43AV10084
Primer mix5 uM each14AV10045
TopTaq5 U/ul0.054.855AV10044
Milli Q5.75557.756AV10034
10 ul7AV10027
8AV10025
PCR cycle
94C3 minNo.
61Pennsylvania (PA) B3
98C10 sec62Ithaca 1 C3
60C3 min63Ithaca 1 C6
x3564Harvard A4
65Ithaca 1 B4
72C5 min66Ithaca 1 C5
67Baltimore B2
68Baltimore B3
69Baltimore B6
70E. Windsor C3
71Pennsylvania (PA) D6
72Baltimore B5


loaded 2.0ul of each sample

Image:0049.jpg

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