User:Torsten Waldminghaus/Methylation analysis with qPCR: Difference between revisions
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(New page: *As independent method and first step one could analyse cutting by methylation sensitive REN with qPCR **on whole chromosome **clusters and isolated GATCs **coding regions and intergenic...) |
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This protocoll describes methylation analyse by cutting bwith methylation sensitive REN followed by qPCR | |||
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| HphI || GGTGA || No star activity; reaction at 37°C; heatinactivation at 20 min 65°C||http://www.fermentas.com/catalog/re/hphi.htm | | HphI || GGTGA || No star activity; reaction at 37°C; heatinactivation at 20 min 65°C||http://www.fermentas.com/catalog/re/hphi.htm | ||
|- | |- | ||
|} | |} | ||
*'''HphI''' seems to be a good choice since it can be heatinactivated and has no star activity (does not cleave unspecific when DNA is overdigested). | *'''HphI''' seems to be a good choice since it can be heatinactivated and has no star activity (does not cleave unspecific when DNA is overdigested). | ||
*In unsynchronized cultures the detection of hemimethylation will be difficult. If every GATC is hemimethylated for about 1 min and replication of the chromosome takes about 50 min, than about 2% of a specific GATC will be in a hemimethylated state giving only 1% cut with an overlapping enzyme. | *In unsynchronized cultures the detection of hemimethylation will be difficult. If every GATC is hemimethylated for about 1 min and replication of the chromosome takes about 50 min, than about 2% of a specific GATC will be in a hemimethylated state giving only 1% cut with an overlapping enzyme. However, some GATCs stay hemimethylated much longer as for example in oriC, dnaA or the GATC-cluster (ref.) | ||
==Procedure== | |||
*Isolate DNA as for example in dnaC2 synchronized cells ([[User:Torsten Waldminghaus/Notebook/dnaC2 temperature shift experiment | link to protocol]]) | |||
===DNA digest=== | |||
*Dilute 500 ng DNA in 17 μL dH<sub>2</sub>O | |||
*add 2 μL of 10x restriction buffer (NEB Buffer 4) | |||
*split in 2 x 9.5 μL | |||
*add 0.5 μL HphI to one tube and 0.5 μL 50% glycerole to the other tube | |||
*incubate at 37°C for 1 h | |||
*incubate at 65°C for 20 min to denature HphI | |||
*add 90 μL dH<sub>2</sub>O to each tube mix and spin down | |||
*use 5 μL per qPCR reaction (corresponding to 15 ng) | |||
===qPCR=== | |||
Revision as of 13:46, 17 February 2009
This protocoll describes methylation analyse by cutting bwith methylation sensitive REN followed by qPCR
| Enzyme | Site | Notes | Link |
|---|---|---|---|
| HphI | GGTGA | No star activity; reaction at 37°C; heatinactivation at 20 min 65°C | http://www.fermentas.com/catalog/re/hphi.htm |
- HphI seems to be a good choice since it can be heatinactivated and has no star activity (does not cleave unspecific when DNA is overdigested).
- In unsynchronized cultures the detection of hemimethylation will be difficult. If every GATC is hemimethylated for about 1 min and replication of the chromosome takes about 50 min, than about 2% of a specific GATC will be in a hemimethylated state giving only 1% cut with an overlapping enzyme. However, some GATCs stay hemimethylated much longer as for example in oriC, dnaA or the GATC-cluster (ref.)
Procedure
- Isolate DNA as for example in dnaC2 synchronized cells ( link to protocol)
DNA digest
- Dilute 500 ng DNA in 17 μL dH2O
- add 2 μL of 10x restriction buffer (NEB Buffer 4)
- split in 2 x 9.5 μL
- add 0.5 μL HphI to one tube and 0.5 μL 50% glycerole to the other tube
- incubate at 37°C for 1 h
- incubate at 65°C for 20 min to denature HphI
- add 90 μL dH2O to each tube mix and spin down
- use 5 μL per qPCR reaction (corresponding to 15 ng)
