Jie:LabNotes/CpgSeq/2009-7-25
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DNA and RNA extraction of BJ with Qiagen Allprep DNA/RNA Mini kit[edit]
sample | DNA concentration | DNA 260/280;260/230 | RNA concentration | RNA 260/280;260/230 |
BJ | 56.2ng/ul x 100ul | 2.11/1.81 | 465.2ng/ul x 30ul | 2.1/2.2 |
PGP1L | 16.4ng/ul x 50ul | 2.17/0.21 | 65.7ng/ul x 30ul | 2.02/0.61 |
ASM validation of GLIPR1L2 of BJ and BJiPS12[edit]
bisufite conversion of BJ gDNA[edit]
I used the zymo kit to do the bisulfite conversion of BJ gDNA. 20ul gDNA(56.2ng/ul) -> 9ul bis_gDNA(230ng/ul).
I suspect that the RNA contamination exists in BJ gDNA so that the quantification after bisulfite conversion is higher than original amount.
PCR primer of GLIPR1L2 refer to LabNotes on [1]
2x iQ supermix 50 Primer F (100uM) 0.4 Primer R (100uM) 0.4 bisulfite DNA: 0.1 dH2O 50 total 100ul 96C 3min-> 40x (95C 30sec, 62C 1min, 72C 1min)-> 72C 5min.
genomic alleotype validatioon of GLIPR1L2 on BJ and BJiPS12[edit]
reaction system x2 H2O 25ul 50ul 2x Mastermix 25ul 50ul AmpF(10uM) 0.2ul 0.4ul AmpR(10uM) 0.2ul 0.4ul template 1ul/each from each sample Total 50ul 300ul 94C 2min -> (94C 45S -> 55C 45S -> 72C 45S) x 35 ->72C 3 min -> 15C hold.
primers: refer to LabNotes on [2]
ASE validation of GLIPR1L2 of BJ and BJiPS12; ASE validation of MMp9 of PGP1L and PGP1F[edit]
Making cDNA of BJ, PGP1L, PGP1F, BJiPS12[edit]
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[edit]
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[edit]
The primers and PCR conditions had been optimized on 09-06-29. refer to LabNotes on [3] 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.
File:20090726 BJ genotype ASM ASE.jpg20090726_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.
results of genotyping and ASE of BJ and BJiPS12[edit]
The geneotype of BJ is C/T. The cDNA showed C allele specific expression of BJ and BJiPS12.