User:Torsten Waldminghaus/Methylation analysis with qPCR

This protocoll describes methylation analyse by cutting bwith methylation sensitive REN followed by qPCR


 * 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 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):


 * 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