User:Torsten Waldminghaus/Methylation analysis with qPCR: Difference between revisions
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==Procedure== | ==Procedure== | ||
*Isolate DNA as for example in dnaC2 synchronized cells ([[User:Torsten Waldminghaus/Notebook/dnaC2 temperature shift experiment | link to protocol]]) | *Isolate DNA as for example in dnaC2 synchronized cells ([[User:Torsten Waldminghaus/Notebook/dnaC2 temperature shift experiment |link to protocol]]) | ||
===DNA digest=== | ===DNA digest=== | ||
| Line 25: | Line 25: | ||
===qPCR=== | ===qPCR=== | ||
*to quantify the ammount of restriction and with that the ammount of methylation one can use qPCR with specific primers | |||
*primers need to border a region containing only the HphI site to be analyzed (note that HphI cuts 8 or 9 bp away from recognition site!) | |||
*as two controls one should include one set of primers for a region without any HphI site (for example datA primers) and a set of primers with a HphI site not overlapping a GATC (for example uvrD) | |||
*for primers refer to [[User:Torsten Waldminghaus/Primers]] | |||
*for each primer set 3 PCR reactions should be prepared for each DNA (triplicate) | |||
*calculate how many reactions for each primer set you need and mix a corresponding master mix containing the following (include 1-2 reactions extra in the calculation to be sure you have enough): | |||
{| border="3" | |||
! component!! for 1 reaction (μL) | |||
|- | |||
| TaqManMix || 12.5 | |||
|- | |||
|- | |||
| Primer fw || 2.25 | |||
|- | |||
|- | |||
| Primer rv || 2.25 | |||
|- | |||
|- | |||
| Probe || 0.625 | |||
|- | |||
|- | |||
| dH<sub>2</sub>O || 2.375 | |||
|- | |||
|} | |||
*'''Note''': The probe is fluoreszens labeld and light sensitive so you should not let your stock lay arround. Pipetting without turning out all the light is however no problem. | |||
Revision as of 14:08, 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)
qPCR
- to quantify the ammount of restriction and with that the ammount of methylation one can use qPCR with specific primers
- primers need to border a region containing only the HphI site to be analyzed (note that HphI cuts 8 or 9 bp away from recognition site!)
- as two controls one should include one set of primers for a region without any HphI site (for example datA primers) and a set of primers with a HphI site not overlapping a GATC (for example uvrD)
- for primers refer to User:Torsten Waldminghaus/Primers
- for each primer set 3 PCR reactions should be prepared for each DNA (triplicate)
- calculate how many reactions for each primer set you need and mix a corresponding master mix containing the following (include 1-2 reactions extra in the calculation to be sure you have enough):
| component | for 1 reaction (μL) |
|---|---|
| TaqManMix | 12.5 |
| Primer fw | 2.25 |
| Primer rv | 2.25 |
| Probe | 0.625 |
| dH2O | 2.375 |
- Note: The probe is fluoreszens labeld and light sensitive so you should not let your stock lay arround. Pipetting without turning out all the light is however no problem.
