Difference between revisions of "C-Fos Methylation"

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(→‎Rat c-fos Primers: added our current primers)
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==Rat c-fos Primers used by Alcala and Houpt==
 
==Rat c-fos Primers used by Alcala and Houpt==
  
 +
Primers designed with Primer 3 using the 300 bases upstream of c-Fos transcription start based on gi|62750806|ref|NC_005105.2|NC_005105
 +
(equivalent to bases 172-471 of U02631, which claims start of 5’UTR is at 455.)
  
Forward:
+
Forward at -261 (cFosSense-261):
 +
:5'-CCATCCCCGAAATCCTACAT-3'
  
Reverse:
+
Reverse at -6 (cFosAS-6):
 +
:5'-CAATCGCGGTTGGAGTAGTA-3'
  
Product size:
+
Product size: 256 bp
  
 
PCR conditions for use with HotStarTaq mix (progam CFOSGENO on thermocycler)
 
PCR conditions for use with HotStarTaq mix (progam CFOSGENO on thermocycler)
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4° for overnight
 
4° for overnight
 
  
 
==Rat c-fos Primers from Literature==
 
==Rat c-fos Primers from Literature==

Revision as of 17:23, 23 January 2009

DNA Methylation sites in c-Fos promoter

Normal Rat Liver, From Shimizu et al., Cancer Sci 2007; 98: 1318–1322


Approximate MW:

38,337 daltons (unmethylated)

38,252 daltons (methylated)

each methylation site causes 15 dalton decrease in MW 

Rat c-fos Primers used by Alcala and Houpt

Primers designed with Primer 3 using the 300 bases upstream of c-Fos transcription start based on gi|62750806|ref|NC_005105.2|NC_005105 (equivalent to bases 172-471 of U02631, which claims start of 5’UTR is at 455.)

Forward at -261 (cFosSense-261):

5'-CCATCCCCGAAATCCTACAT-3'

Reverse at -6 (cFosAS-6):

5'-CAATCGCGGTTGGAGTAGTA-3'

Product size: 256 bp

PCR conditions for use with HotStarTaq mix (progam CFOSGENO on thermocycler)

95° for 15 min to activate Taq

Repeat 35 times:

94° for 1 min
59° for 1 min
72° for 1 min
72° for 10 min final extension

4° for overnight

Rat c-fos Primers from Literature

From Shimizu et al., Cancer Sci 2007; 98: 1318–1322

Bisulfite sequencing (Genomic DNA)

GenBank AF126534

F: 5′-TATTTATAGGTGAAAGTTATAGATTG-3′

R: 5′-CACTAATAAAAACTACAAAACAAAACT-3′

Annealing temperature 54

Product size 292 bases

4-23-08 -- could NOT confirm with blast -- need to check earlier references


RT-PCR (from RNA)

GenBank X06769

F: 5′-TTGCGCAGATCTGTCCGTCT-3′

R: 5′-GTTGATCTGTCTCCGCTTGG-3′

Annealing temperature 65

4-23-08 -- confirmed with blast