Jie:LabNotes/CpgSeq/2009-7-2: Difference between revisions

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I purified the amplicons and selected 37 amplicons from 14 genes to Genewiz to do the sanger sequencing on 20090709.
I purified the amplicons and selected 37 amplicons from 14 genes to Genewiz to do the sanger sequencing on 20090709.
The plate label: [[Media: 20090709_plate_to_Genewiz_for_ASE_valid.xls]]20090709_plate to Genewiz for ASE valid
The plate label: [[Media: 20090709_plate_to_Genewiz_for_ASE_valid.xls]]20090709_plate to Genewiz for ASE valid
===results of ASE sanger sequencing results===
I got high quality reads for 10 genes.
ATF7IP, C20orf96, PAN2, PRIC285, DNMT3B, LSP1 are not imprinted genes. No ASE were demonstrated either.
CPNE8: No65: T; No75 C/T. (I did not do No74 yet. The Hapmap database show that No74 should be CC. However, if it is imprinted gene, father No75 should demonstrate monoallelic expression as well. I need to do the No74 ASE)
 
COX412: need resequence using forward primer;
GGT7: need to redo No64.
GLIPR1L2: No64 is C, No 72 is T, No 73 is C. This is a imprinted gene candidate. I need to repeat the No64 and do DNA sequencing validation.
KIAA1853: need to purify the amplicon and redo the sequencing.
MATN4: need to redo the No65, 74,75 ASE.
c20orf85: need to redo the No64, 72, 73 ASE.

Revision as of 19:07, 10 July 2009

DNA and RNA extraction from samples and ASE validation for fuzzlmethylated genes

DNA and RNA extraction with Qiagen Allprep DNA/RNA Mini kit

sample DNA concentration DNA 260/280;260/230 RNA concentration RNA 260/280;260/230
GM12864 94.1ng/ul x 100ul 1.97/0.17 1515.7ng/ul x 30ul 2.1/2.2
GM12865 130.9ng/ul/ul x 100ul 1.98/1.44 1515.9ng/ul x 30ul 2.11/2.19
GM12872 217.8ng/ul x 100ul 1.94/1.83 1638ng/ul x 30ul 2.1/2.18
GM12873 82.6ng/ul x 100ul 1.92/1.49 1416.4ng/ul x 30ul 2.11/2.14
GM12875 140ng/ul x 100ul 1.89/1.03 838.3ng/ul x 30ul 2.13/2.11

Making cDNA

                          64        65     72    73     75    
  RNA                     3ul       3ul    3ul   3ul    4ul
  10X DNase I buffer      5ul       5ul    5ul   5ul    5ul
  RNase-Free DNase I      2.5ul   2.5ul   2.5ul  2.5    2.5
  H2O                    39.5ul   39.5ul  32.5ul 32.5   31.5
  37C 10min 
 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;
Reverse transcription
                 64~75      
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:
GM12864: 74.8ng/ul; 2.17/1.16;
GM12865: 72.3ng/ul; 2.16/1.09;
GM12872: 79.1ng/ul; 2.08/0.78;
GM12873: 76.4ng/ul; 2.16/1.17;
GM12875: 66.4ng/ul; 2.17/1.14;

PCR to validat the ASE

 The primers and PCR conditions had been optimized on 09-06-29. refer to LabNotes on [1] 
                                            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
For 12864/12872/12873: A1, C1-E1, A3-C3, E3-F3, H3, A5.
For 12865/12875: G1, H1, D3.
The annealing temperature for A5 and H1 is 58C. Annealing Temperature for others are 62C.
96C 3min-> 37x (95C 30sec, 58C or 62C 1min, 72C 1min)-> 72C 5min.

File:20090704 ASE valid 1.jpg20090704_ASE_valid_1

File:20090704 ASE valid 2.jpg20090704_ASE_valid_2

I repeated the amplification of B1, D1-H1, A3-B3, D3, H3 with 42 PCR cycles.

File:20090706 ASE valid 3.jpg20090706_ASE_valid_3

File:20090706 ASE valid 4.jpg20090706_ASE_valid_4

I purified the amplicons and selected 37 amplicons from 14 genes to Genewiz to do the sanger sequencing on 20090709. The plate label: Media: 20090709_plate_to_Genewiz_for_ASE_valid.xls20090709_plate to Genewiz for ASE valid

results of ASE sanger sequencing results

I got high quality reads for 10 genes.
ATF7IP, C20orf96, PAN2, PRIC285, DNMT3B, LSP1 are not imprinted genes. No ASE were demonstrated either.

CPNE8: No65: T; No75 C/T. (I did not do No74 yet. The Hapmap database show that No74 should be CC. However, if it is imprinted gene, father No75 should demonstrate monoallelic expression as well. I need to do the No74 ASE)
 
COX412: need resequence using forward primer;

GGT7: need to redo No64.

GLIPR1L2: No64 is C, No 72 is T, No 73 is C. This is a imprinted gene candidate. I need to repeat the No64 and do DNA sequencing validation.
KIAA1853: need to purify the amplicon and redo the sequencing.
MATN4: need to redo the No65, 74,75 ASE.
c20orf85: need to redo the No64, 72, 73 ASE.