User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/04/09

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Contents

Individual Rotifer Extraction fresh from Moss sample

1) Soak fresh Moss sample in petri dish with autoclaved/filtered spring water - enough to cover sample. 2) Wait 1 hour and/or move sample around in water. 3) Search for rotifers. Maybe helpful to remove moss to search. 4) Onto a petri-dish, place 1 rotifer in 50ul of autoclaved/filtered spring water. Rinse 3 to 5 times with fresh autoclaved/filtered spring water, taking care not to suck up rotifer. Attempt to remove all debris leaving only single rotifer. 5) Pick rotifer with ~50ul of clean autoclaved/filtered spring water and place in extraction tube. 6) Proceed with Instagene extraction.

Reaction mixture conc. 1x 10 ' ' AMP Lane Template
Template Instagene?11IRP 1
TopTaq buffer10x1102IRP 2
dNTP mix10 mM each0.223IRP 3
Primer mix5 uM each0.554IRP 4
TopTaq (QIAGEN)5U/ul0.050.55IRP 5
Milli Q water7.2572.56IRP 6
10 ul909.0ul / RXN7Postitive Control CCA3
*Control AV 1ng/ul8Negative Control
PCR cycle
94C3 min
94C10 sec
45-68C3 min
RAMP 0.5C/sec
x35
68C5 min

Loaded 2.5ul PCR product

image:09April10.tif

LCO/HCO

Reaction mixture conc. 1x 19 ' ' AMP Lane Template '
Template Instagene?1ABostonA1
TopTaq buffer10x119BBostonA4
dNTP mix10 mM each0.23.8CCape Cod (CC)A1
Primer mix5 uM each119DWoods Hole 3A5
TopTaq (QIAGEN)5U/ul0.050.95IWoods Hole 3C2
Milli Q water6.75128.2575BaltimoreA5
10 ul1719.0ul / RXN76Pennsylvania (PA)B1
*Control AV 1ng/ul+Positive Control AV
-Negative Control
PCR cycle
94C3 min
94C10 sec
45-68C3 min
RAMP 0.5C/sec
x35
68C5 min


Loaded 2.5ul PCR product

image:09April10.tif


Alien Genes New Primer Screen

Reaction mixture conc. 1x x16.5 ' lane '
Temp??227Ithaca 2 A3
TopTaq Buffer10x116.528Ithaca 2 A4
dNTP mix10 mM each0.23.329Ithaca 2 A5
Primer mix (HIS F/R1)5 uM each116.537Tsukuba 3 D2
TopTaq5 U/ul0.050.82540Woods Hole 1 A6
Milli Q5.7594.87543Yokohama B5
10 ul47Harvard A1
48Ithaca 2 A6
PCR cycle50Tsukuba 1 B5
94C3 min51Yokohama A2
55Yokohama B2
98C10 sec56Yokohama A4
60C3 min61Pennsylvania(PA) B3
x3562Ithaca1 C3
75Baltimore A5
72C5 min76Pennsylvania (PA) B1

image:0070-2.jpg

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