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

• 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.

• Make a drawing of the 96 well PCR plate with the location of your primer sets and DNAs
• Pipette in 20 μL of the master mix in the designated well (with one pipette tip per master mix)
• Add 5 μL of the DNA to the corresponding well using a new tip for every 5 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