User:Torsten Waldminghaus/Methylation analysis with qPCR

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

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)

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 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