Jie:LabNotes/CpgSeq/2009-7-27: Difference between revisions

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>Jie deng
(New page: ==MMP9 ASE of PGP1F and PGP1L== ===PCR=== The primers x 6 2x iQ supermix 40 240 Primer F (100uM) ...)
 
>Jie deng
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==MMP9 ASE of PGP1F and PGP1L==
==MMP9 ASE of PGP1F and PGP1L==
The cDNA of PGP1F and PGP1L: refer to LabNotes [http://genome-tech.ucsd.edu/LabNotes/index.php/Jie:LabNotes/CpgSeq/2009-7-25]
===PCR===
===PCR===
 
I designed the primer according to the alleotyping of PGP1. The target cover the rs13969(C/A) on exon.
  The primers  
  The primers: [[Media: 20090727_MMP9_ASE.xls|MMP9_ASE_primer]]
                                             x 6
                                             x 4
  2x iQ supermix                40          240          
  2x iQ supermix                40          160          
  Primer F (100uM)            0.4          2.4 each
  Primer F (100uM)            0.4          1.6 each
  Primer R (100uM)            0.4          2.4 each
  Primer R (100uM)            0.4          1.6 each
  cDNA samples as above:      0.1  
  cDNA samples as above:      0.1  
  dH2O                          40          240
  dH2O                          40          160
  total                        80ul
  total                        80ul


96C 3min-> 45x (95C 30sec, 58C or 62C 1min, 72C 1min)-> 72C 5min.
96C 3min-> 45x (95C 30sec, 58C or 62C 1min, 72C 1min)-> 72C 5min.
[[Image:20090727_PGP1F_PGP1L_MMP9_ASE.jpg|300px]]20090727_PGP1F_PGP1L_MMP9_ASE
I sent PGP1F_62C and PGP1L_58C to do the sanger sequencing.
The sanger sequencing results did not show ASE of MMP9 in two cell lines.

Latest revision as of 18:49, 28 July 2009

MMP9 ASE of PGP1F and PGP1L[edit]

The cDNA of PGP1F and PGP1L: refer to LabNotes [1]

PCR[edit]

I designed the primer according to the alleotyping of PGP1. The target cover the rs13969(C/A) on exon.
The primers: MMP9_ASE_primer
                                           x 4
2x iQ supermix                40           160         
Primer F (100uM)             0.4           1.6 each
Primer R (100uM)             0.4           1.6 each
cDNA samples as above:       0.1	  	
dH2O                          40           160
total                         80ul

96C 3min-> 45x (95C 30sec, 58C or 62C 1min, 72C 1min)-> 72C 5min.

File:20090727 PGP1F PGP1L MMP9 ASE.jpg20090727_PGP1F_PGP1L_MMP9_ASE

I sent PGP1F_62C and PGP1L_58C to do the sanger sequencing.
The sanger sequencing results did not show ASE of MMP9 in two cell lines.