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==ASE validation of GLIPR1L2 of BJ and BJiPS12; ASE validation of MMp9 of PGP1L and PGP1F== ===Making cDNA of BJ, PGP1L, PGP1F, BJiPS12=== BJ PGP1L PGP1F(122ng/ul from Kun) BJiPS12(149ng/ul from Kun) RNA 10ul 30ul 30ul 20ul 10X DNase I buffer 5ul 5ul 5ul 5ul RNase-Free DNase I 2.5ul 2.5ul 2.5 2.5 H2O 32.5ul 12.5ul 12.5 20.5 37C 15min Clean up and concentrate all RNAs with Zymo DNA-Free RNA kit * Add 4 volumes of RNA binding buffer; * Transfer to Zymo-spin columns in collection tubes, spin @15k rpm for 30s; * Add 200ul RNA Wash buffer, spin @15k rpm for 30s; * Add 10ul DNase/RNase-free water, spin @15k rpm for 30s; Bu sure that PFP1F and BJiPS12 were correctly labelled. ===Reverse transcription=== RNA 8ul 10mM dNTP 1ul 50uM dT12-18 1ul 65C 5min -> chill on ice for 1min Add 10X RT buffer 2ul 25mM MgCl2 4ul 0.1M DTT 2ul RNaseOUT 1ul Superscript III 1ul Incubate all tube at 50C for 50min. Cleanup the first-strand reactions with qiaquick columns. quantify: BJ:88.5ng/ul; 2.14/1.48 PGP1L:33.8ng/ul;2.23/1.48 PGP1F:70.6ng/ul;2.15/1.57 BJiPS12:59.4ng/ul;2.17/1.37 ===PCR to validat the ASE of GLIPR1L2 on BJ and BJiPS12=== The primers and PCR conditions had been optimized on 09-06-29. refer to LabNotes on [http://genome-tech.ucsd.edu/LabNotes/index.php/Jie:LabNotes/CpgSeq/2009-6-29] The primer for GLIPR1L2 is B3. x 40 2x iQ supermix 20 800 Primer F (25uM) 0.5 0.5 each Primer R (25uM) 0.5 0.5 each cDNA samples as above: 0.1 dH2O 20 800 total 40ul 96C 3min-> 45x (95C 30sec, 64C 1min, 72C 1min)-> 72C 5min. I got the BJ_genotype, ASE of BJ, PGP1F and BJiPS12. However, I did not get the BJ_ASM. [[Image:20090726_BJ_genotype_ASM_ASE.jpg]]20090726_BJ_genotype_ASM_ASE I column purified the BJ_genotype and do sanger sequencing. I agrose gel purified the BJ, PGP1F and BJiPS12 ASE and do sanger sequencing. I redo the bisulfite conversion of BJ and ASM of BJ.
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