User:Alexander S Mikheyev/Notebook/rotifer alien genes/2009/11/18

Alien genes

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To do restriction digest of A. vaga amp AV genes Primer sequences for A vaga alien genes identified by Gladyshev the last two pairs amplify a 3kb intergenic fragment in one of the collinear pairs (hsp82 region) Well Seq Name Seq Length Tm A01 AV10158_F ATTAGCTTATGCAACGTCCAAAACA 25 63 A02 AV10158_R GCGGAGGATAATTTTCGTTGTTATC 25 63 A03 AV10153_F GAGCTGGTGAAATATTCATGATTCG 25 63 A04 AV10153_R ATAATCCCAAAATGCTTGTCCACTT 25 63 A05 AV10151_F TCACCCCAAGGAGCGTGTATAG 22 63.5 A06 AV10151_R GTGGTACTCTCTGGACACGCGATA 24 64.8 A07 AV10150_F GTGTGTATGCCTCTTTTCCATTCAC 25 63 A08 AV10150_R ACCATGTAAGGAGGGATTGAATCAT 25 63 A09 AV10140_F AAATGCATCATCAGCAATTAAAGGA 25 62.9 A10 AV10140_R CGTGTTTGATCTTTCGCTACATACC 25 63.1 A11 AV10134_F TCTATGGTAGCACTTGACGGAAGAC 25 62.9 A12 AV10134_R TTGGTCCAGCAATATAACAATCCAC 25 63.1 B01 AV10130_F TGGTGAAAATCAAGAAGGAGATGAA 25 63.2 B02 AV10130_R TTGACCAACAAATTTTCTACCAGGA 25 62.9 B03 AV10126_F AAAAGATGGTGCAATGTTAGCAAAA 25 63 B04 AV10126_R TCGTTCACGTATATCTCCTGGAAAA 25 63 B05 AV10123_F CAATATCGTTCAAGTCACCCTTCAC 25 63 B06 AV10123_R CTTGCCGAAAATCTATGTTATCACG 25 63 B07 AV10121_F ATCGATCTTTTATTCGCAAATCCAT 25 63 B08 AV10121_R TACCAACCGATTCTATGACGAAAGA 25 63 B09 AV10104_F ACACTTGAAGATCAACCTGCATTTC 25 62.8 B10 AV10104_R CGGAAATTAATTGTGCAGACATAGC 25 63 B11 AV10109_F ACAAGTGGTCGACAAGGTTTTTGTA 25 63.1 B12 AV10109_R CGGTTTTAAGTCTGGACTACCATCA 25 62.6 C01 AV10099_F TCCAAACGAGCAATACCATTAT 22 58.5 C02 AV10099_R GGTGGATCTGGTAATATTGCTT 22 57.2 C03 AV10098_F GCTGAAAGTTCTTCATCGAAATCAA 25 62.9 C04 AV10098_R GAAGAGCAAGTTGTGGATCATCATT 25 62.8 C05 AV10094_F ATGCTGGTCCATGTAAAATGACACT 25 63 C06 AV10094_R TTTCCATACTCCAAGCCAAATAGGT 25 63.1 C07 AV10092_F TCTTTCACAATTTCTTCCCAATCAA 25 63 C08 AV10092_R TTCATCACCGACTAATGATTGTCCT 25 63 C09 AV10089_F CCATTAGGAGCTCTTGTTTGTGCTA 25 63 C10 AV10089_R TGATCTAATGCATCTGCAAAATCCT 25 63.1 C11 AV10084_F GGTTGATTGTAATGATGTGGGAAAA 25 63 C12 AV10084_R TATTACATGTTAAATCCGCCCAATG 25 62.9 D01 AV10082_F GGTTCAACATGTAAACAATGTGCTG 25 62.7 D02 AV10082_R ACGTGGCAGCTTGACAAATACTATC 25 62.8 D03 AV10075_F GAACGATGAACGGTTTTAGTCCTCT 25 63 D04 AV10075_R CACCATATGCATCTCCACCAATAAT 25 63 D05 AV10071_F AGACCGCCAAATAAGCTTGTAACTC 25 63 D06 AV10071_R TAATGGCAAAAACCTCACGTTAGAA 25 63 D07 AV10045_F CAGGTGAAGATGGTCAAATACAAGG 25 63 D08 AV10045_R CAATGGATGGTGAAACGTGAATAAT 25 63 D09 AV10044_F ATCTGACCGATGTTCCAGTTGATAA 25 63 D10 AV10044_R TTCATCCTCGATATACACACGCTTT 25 63.1 D11 AV10043_F GGCTCGATCCACTTCTTTATCCTAA 25 63 D12 AV10043_R TTGAGTTAAATGACGAATCCATGCT 25 63 E01 AV10042_F ACACATACCGATTTTCACGTGCTAT 25 62.9 E02 AV10042_R AAGAAGATCACTATGACAGGCAACG 25 62.9 E03 AV10041_F TACCAATATTTGTGCTCCAGCTGTT 25 63.1 E04 AV10041_R ACCGTGGTGTATTCAAACTACCAGA 25 62.9 E05 AV10034_F CAGTCTTTCGAATCTTGGGTACAGA 25 62.9 E06 AV10034_R GCCGTATATGTTATGAGCTTGATCG 25 63 E07 AV10028_F TGATATTTATCCTCCGGCAAAGAAT 25 63 E08 AV10028_R GAAGAATATCTGCCAACTTCGGATT 25 62.9 E09 AV10027_F TACTACCATGTCCAGCTCCATTCAT 25 63 E10 AV10027_R ATATTATTTGCAATATGCGGCTCCT 25 63 E11 AV10025_F ATACCATCTCTTCAATGTCCACGAA 25 63 E12 AV10025_R ATATGGTACGTCACTGCCGAATAGA 25 63 F01 AV10016_F TTAAATCTGTGCAATACCACCATCC 25 63.1 F02 AV10016_R AGGACCAATCGATACAGCAATGTTA 25 62.9 F03 AV10011_F ATCACCTTGCACTGCTGTTACATTT 25 63.2 F04 AV10011_R TTGATTACAGGTGGTTCAGAGGGTA 25 63 F05 AV10007_F GTCCTCGTAGCAGATATCAATGGTG 25 63.1 F06 AV10007_R TTTAAATCACCCACATCAATCCAAG 25 63.1 F07 AV10002_F AATTGGATCTGTGGAAAGACATTCA 25 63 F08 AV10002_R CACTATAACACTTGCCCTTGAAACG 25 63.1 F09 5475_F CCNATNGAACCNTTTAGTCGATGTT 25 63.4 actually located at 27927 F10 8047_R CATTCNGCTTGTGTTGGATAGAGAC 25 63.2 actually located at 30146 F11 4220_F GCTCGTTCATCNCCACAAGATAAAT 25 64 F12 7822_R TTGTGTTGGATAGAGACCAAGNGA 24 62.5 looking for alien genes in vaga and ricciae Reagents 1x conc. 40 h2o 7.75 - 230 10x 1 10x 40 dye 1 10x 40 dNTP 0.2 10 mM 8 Forward 1 5 uM ea Taq 0.05 5 U/um 2 DNA 1 20 ng/ul 40 TOTAL 10 Cocktail per vial 9 3m 94C (10s 94C; 3m 60C)x35 made 2 big 1% gels with 80ml TAE loaded 2ul vaga followed by ricciae, left to right maybe try an RsaI or Alu1 digest of long fragment for kicks sequence AV10016 (6th from end) and AV10027 (8th from end) both located on EU643475[accn] trying restriction enzymes on A. vaga and ricciae water 3 21 enzyme 1 NEB 2 1 7 DNA 5 35 BamHI EcoRI MspI Tsp509i BamHi/MspI BamHI/Tsp509i EcoRI/MspI 37C for 2 hours vaga, then ricciae in the following order: BamHI EcoRI MspI Tsp509i BamHi/MspI BamHI/Tsp509i EcoRI/MspI