User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/03/31

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Testing fusion primers

  • Testing whether fusion primers for genes that were amplified on the 26th work correctly. Each forward primer has an amplicon A adapter, a barcode and the rotifer primer. Each reverse primer has a B adapter and a rotifer primer.

PCR conditions

Reaction mixture conc. 1x 7
water 7.25 50.75
TopTaq buffer 10x 1 7
dNTP mix 10 mM each 0.2 1.4
Primer mix 5 uM each 1
TopTaq (QIAGEN) 5U/ul 0.05 0.35
template 20 ng/ul 0.5 0.5
9
  • 95C 3 min (60C 1 min, 95C 5s)x35
  • the histone gene, which is about 600 to 800 bp was amplified with a 50:50 mixture of forward and reverse fusion primers (i.e., so that it can be sequenced from both ends).

gel

Asm100331.png

  • maybe it makes sense to increase annealing temperature to 35C after a few cycles?

SNARF-1 and RED Rotifer Staining

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

TREATMENT

Test 200ul of 1) 1:1000 dye dilution 2) 1:1200 dye dilution 3) 1:1400 dye dilution all treated with dye for 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 autoclaved 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 autoclaved spring water. Repeat 3-5 times.

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

(7) Visualize in the GFP channel of a fluorescent microscope.

RESULTS

Snarf-1 1) 1:1000 dye dilution - alive stained 2) 1:1200 dye dilution - alive stained 3) 1:1400 dye dilution - alive stained

Red 1) 1:1000 dye dilution - weak stain 2) 1:1200 dye dilution - weak stain 3) 1:1400 dye dilution - weak stain

8 days later

Snarf-1 - Rotifers still alive and stained, but lots of background. Probably due to stained food being consumed and waste accumulation. 1:1000 best

Testing of extracted genome

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 49 Pennsylvania (PA) D4
94C 3 min 50 Tsukuba 1 B5
51 Yokohama A2
98C 10 sec 55 Yokohama B2
60C 3 min AV Adineta vaga
x35
72C 5 min


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