Jie:LabNotes/CpgSeq/2009-7-25

From ZhangLabWiki
Jump to navigation Jump to search

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.