Difference between revisions of "C-Fos Methylation"
(→Rat c-fos Primers: added our current primers) |
m (added lowercase title) |
||
| (3 intermediate revisions by the same user not shown) | |||
| Line 1: | Line 1: | ||
| + | {{lowercase title}} | ||
| + | |||
==DNA Methylation sites in c-Fos promoter== | ==DNA Methylation sites in c-Fos promoter== | ||
| Line 16: | Line 18: | ||
==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) | ||
| Line 39: | Line 45: | ||
4° for overnight | 4° for overnight | ||
| + | Sequenced PCR product from Brain and Tail amplification on 1/29/09 | ||
| + | |||
| + | Matched AY786174.1 base pairs 85-339 | ||
| + | |||
| + | ccatcc ccgaaatcct acatgcggag ggtccaggag accttctaag atcccaattg tgaacactca taggtgaaag | ||
| + | ttacagactg agacgggggt tgagagcctg gggcgtagag ttgatgacag ggagcccgca gagggcattc | ||
| + | gggagcgctt tcccccctcc agtttctctg ttccgctcat gacgtagtaa gccattcaag cgcttctata aagcggccag | ||
| + | ctgaggcgcc tactactcca accgcgatt | ||
==Rat c-fos Primers from Literature== | ==Rat c-fos Primers from Literature== | ||
| Line 70: | Line 84: | ||
''4-23-08 -- confirmed with blast'' | ''4-23-08 -- confirmed with blast'' | ||
| + | |||
| + | [[Category:Taste Aversion]] | ||
| + | [[Category:c-Fos]] | ||
Latest revision as of 13:14, 5 February 2013
DNA Methylation sites in c-Fos promoter
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
Sequenced PCR product from Brain and Tail amplification on 1/29/09
Matched AY786174.1 base pairs 85-339
ccatcc ccgaaatcct acatgcggag ggtccaggag accttctaag atcccaattg tgaacactca taggtgaaag ttacagactg agacgggggt tgagagcctg gggcgtagag ttgatgacag ggagcccgca gagggcattc gggagcgctt tcccccctcc agtttctctg ttccgctcat gacgtagtaa gccattcaag cgcttctata aagcggccag ctgaggcgcc tactactcca accgcgatt
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