User:Alexander S Mikheyev/Notebook/rotifer alien genes/2010/04/12

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Ethanol Post-PCR Clean Up

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.

Ethanol Clean # MIX Contents ' Size Gene CCA3 Result Ethanol Cleaned ng/ul Sample Type Total Product Remaining bp/ng ul to mix ng added ng/ul
1 23 AV10004 FWD AV10007 RVS 8691 AV212 A good 04Mar10 495.3 DNA x 9ul 0.05698998964446 1.14395581149371 566.601313432836
2 1 AV10007 FWD AV10011 RVS 9262 AV212 A good 04Mar10 437.4 DNA x 9ul 0.047225221334485 1.38049177978421 603.827104477612
3 2 AV10011 FWD AV10016 RVS 11924 AV212 A good 02Mar10 314.9 DNA x 9ul 0.026408923180141 2.4686364304233 777.373611940299
4 24 AV10016 FWD AV10020 RVS 7020 AV212 A good 04Mar10 408.3 DNA x 9ul 0.058162393162393 1.12089661903561 457.662089552239
5 25 AV10020 FWD AV10025 RVS 11202 AV212 A good 04Mar10 403.1 DNA x 9ul 0.035984645599 1.81171799153575 730.30352238806
6 4 AV10025 FWD AV10028 RVS 6843 AV212 A good 04Mar10 330.8 DNA x 9ul 0.048341370743826 1.34861773358119 446.122746268657
7 34 AV10029 FWD AV10034 RVS 12093 AV240 B good 08Mar10 380.5 DNA x 9ul 0.031464483585545 2.07198791848903 788.391402985075
8 40 AV10034 FWD AV10039 RVS 14180 AV240 B good 18Mar10 1128.6 DNA x 9ul 0.079590973201692 0.81911336459648 924.451343283582
9 50 AV10039 FWD AV10041 FWD 19555 AV240 B good 18Mar10 1888.1 DNA x 9ul 0.096553311173613 0.67521278201967 1274.86925373134
10 30 EU643474 FWD EU643474 RVS 10062 AV240 B good 04Mar10 206 DNA x 9ul 0.020473066984695 3.18438023474859 655.982328358209
11 31 AV10045 FWD AV10047 RVS 6762 AV240 B good 04Mar10 393.3 DNA x 9ul 0.058163265306122 1.12087981146897 440.842029850746
12 32 AV10047 FWD AV10049 RVS 12719 AV240 B good 04Mar10 423.1 DNA x 9ul 0.033265193804544 1.95982714647044 829.202865671642
13 33 AV10049 FWD AV10053 RVS 9353 AV240 B good 04Mar10 364.3 DNA x 9ul 0.038950069496418 1.67378468623121 609.75976119403
25 138 AV10083 FWD AV10084.2 RVS 5400 AV TEL L A good 05Apr10 214.1 DNA x 7.5ul 0.039648148148148 1.64431462491373 352.04776119403
14 78 AV10091 FWD AV10092.2 RVS 8127 AV TEL L A good 19Mar10 515.5 DNA x 9ul 0.063430540174726 1.02780190222505 529.831880597015
15 85 AV10092.2 FWD AV10094 RVS 7036 AV TEL L A good 01Apr 1353.9 DNA x 9ul 0.19242467310972 0.33880286138552 458.705194029851
16 16 AV10094 FWD AV10098 RVS 8600 AV TEL L A good 02Mar10 334.4 DNA x 9ul 0.038883720930233 1.67664071984575 560.668656716418
17 134 AV10098 FWD AV10104 FWD 8000 AV TEL L A&B good 01Apr 466.4 DNA x 9ul 0.0583 1.11825094083613 521.55223880597
18 51 AV10104 RVS AV10106 RVS 9618 AV_TEL_L_B good 16Mar 430.3 DNA x 9ul 0.044739030983572 1.45720701627813 627.036179104478
19 111 AV10106 FWD AV10108.2 RVS 13400 AV_TEL_L_B good 29Mar 218.4 DNA x 9ul 0.016298507462687 4 873.6
20 112 AV10107.1 FWD AV10108 RVS 7900 AV TEL L B good 29Mar 214.1 DNA x 9ul 0.027101265822785 2.40557139570713 515.032835820896
21 55 AV10108 FWD AV10111 RVS 14846 AV_TEL_L_B good 16Mar 210.7 DNA x 9ul 0.014192375050519 4.5935954777607 967.870567164179
22 117 AV10111 FWD AV10113 RVS 8400 AV TEL L B good 29Mar 355.8 DNA x 9ul 0.042357142857143 1.5391507890564 547.629850746269
23 128 AV10113 FWD TEL_L_B_3 RVS 11500 AV TEL L B good 01Apr 422.7 DNA x 9ul 0.03675652173913 1.77367244685021 749.731343283582
24 131 AV10114 FWD TEL_L_B_4 RVS 8500 AV TEL L B good 01Apr 374.2 DNA x 9ul 0.044023529411765 1.48089057651348 554.149253731343
16363.2451343284 373.288363700934

Alien Genes New Primer Screen

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 (histone_F1/histone_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 50 Tsukuba 1 B5
94C 3 min 51 Yokohama A2
55 Yokohama B2
98C 10 sec 56 Yokohama A4
60C 3 min 61 Pennsylvania(PA) B3
x35 62 Ithaca1 C3
75 Baltimore A5
72C 5 min 76 Pennsylvania (PA) B1

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