User:Torsten Waldminghaus/Methylation analysis with qPCR: Difference between revisions
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===DNA digest=== | ===DNA digest=== | ||
*Dilute | *Dilute 625 ng DNA in 22.5 μL dH<sub>2</sub>O | ||
*add 2 μL of 10x restriction buffer (NEB Buffer 4) | *add 2.5 μL of 10x restriction buffer (NEB Buffer 4) | ||
* | *take out 2 x 9 μL in new tubes | ||
*add 0.5 μL HphI to one tube and 0.5 μL 50% | *add 0.5 μL HphI (2.5 units) and 0.5 μL EcoRI to one tube and 0.5 μL 50% glycerol and 0.5 μL EcoRI to the other tube | ||
*Note: EcoRI fragments both chromosomal DNAs which is important because one would otherwise compare fragmented (HphI digested) with unfragmented chromosomal DNA (only glycerol) in the qPCR which will influence the result. | |||
*mix by pipetting and short spin in a centrifuge | |||
*incubate at 37°C for 1 h | *incubate at 37°C for 1 h | ||
*incubate at 65°C for 20 min to denature | *incubate at 65°C for 20 min to denature enzymes | ||
*add | *add 190 μL dH<sub>2</sub>O to each tube mix and spin down | ||
*use | *use 10 μL per qPCR reaction (corresponding to 15 ng) | ||
===qPCR=== | ===qPCR=== | ||
| Line 28: | Line 30: | ||
*to quantify the ammount of restriction and with that the ammount of methylation one can use qPCR with specific primers | *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!) | *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 | *as two controls one should include one set of primers for a region without any HphI site (for example uvrD primers) and a set of primers with a HphI site not overlapping a GATC (for example datA) | ||
*for primers refer to [[User:Torsten Waldminghaus/Primers]] | *for primers refer to [[User:Torsten Waldminghaus/Primers]] | ||
*make primer mixes: | |||
{|border="1" | |||
|+ '''Primer Mix''' | |||
!for 250μL | |||
|- | |||
|22.5μL primer fw | |||
|- | |||
|22.5μL primer rv | |||
|- | |||
|6.25μL probe | |||
|- | |||
|198.75μL ddH<sub>2</sub>O | |||
|} | |||
*for each primer set 3 PCR reactions should be prepared for each DNA (triplicate) | *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): | *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): | ||
| Line 39: | Line 55: | ||
|- | |- | ||
|- | |- | ||
| | | primer mix (see above)|| 2.5 | ||
|- | |- | ||
|} | |} | ||
| Line 55: | Line 62: | ||
*Make a drawing of the 96 well PCR plate with the location of your primer sets and DNAs | *Make a drawing of the 96 well PCR plate with the location of your primer sets and DNAs | ||
*Pipette in | *Pipette in 15 μL of the master mix in the designated well (with one pipette tip per master mix) | ||
*Add | *Add 10 μL of the DNA to the corresponding well using a new tip for every 10 μL (wipe of the DNA at the corner of the well when pipetting the DNA into the well) | ||
*cover the 96 well plate with plastic tape cover | *cover the 96 well plate with plastic tape cover | ||
*centrifuge short up to about 1000 rpm | *centrifuge short up to about 1000 rpm | ||
*put in qPCR machine and hope for nice results | *put in qPCR machine and hope for nice results | ||
Latest revision as of 04:19, 20 March 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 625 ng DNA in 22.5 μL dH2O
- add 2.5 μL of 10x restriction buffer (NEB Buffer 4)
- take out 2 x 9 μL in new tubes
- add 0.5 μL HphI (2.5 units) and 0.5 μL EcoRI to one tube and 0.5 μL 50% glycerol and 0.5 μL EcoRI to the other tube
- Note: EcoRI fragments both chromosomal DNAs which is important because one would otherwise compare fragmented (HphI digested) with unfragmented chromosomal DNA (only glycerol) in the qPCR which will influence the result.
- mix by pipetting and short spin in a centrifuge
- incubate at 37°C for 1 h
- incubate at 65°C for 20 min to denature enzymes
- add 190 μL dH2O to each tube mix and spin down
- use 10 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 uvrD primers) and a set of primers with a HphI site not overlapping a GATC (for example datA)
- for primers refer to User:Torsten Waldminghaus/Primers
- make primer mixes:
| for 250μL |
|---|
| 22.5μL primer fw |
| 22.5μL primer rv |
| 6.25μL probe |
| 198.75μL ddH2O |
- 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 mix (see above) | 2.5 |
- 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.
- Make a drawing of the 96 well PCR plate with the location of your primer sets and DNAs
- Pipette in 15 μL of the master mix in the designated well (with one pipette tip per master mix)
- Add 10 μL of the DNA to the corresponding well using a new tip for every 10 μL (wipe of the DNA at the corner of the well when pipetting the DNA into the well)
- cover the 96 well plate with plastic tape cover
- centrifuge short up to about 1000 rpm
- put in qPCR machine and hope for nice results
