Leiden Genome Technology Center (LGTC) - LUMC©
Generation of methylated
amplicons for differential methylation
hybridization
Array protocols
(Judith Boer, Remco van Doorn and Eddy van Roon; last modified 11 April, 2005)
for in house use only
Abbreviations
Purpose
This protocol describes the generation of methylated amplicons from genomic DNA to
screen for differential methylation of genomic regions using e.g. CpG island
microarrays. For labeling and hybridization of amplicons, see "Labeling and
hybridization of genomic amplicons on CpG island microarrays".
Protocol
BOOKING MACHINES
You will need a PCR machine for 0.2 ml thin wall tubes/strips on days 3 (2.5 hrs) and 5 (2
hrs) in the morning.
TIME SCHEDULE (all steps have to be performed in 5 days without freezing)
Day 1 morning: check quality and concentration of DNA; late afternoon: start MseI digest.
Day 2 morning: clean up MseI digest; afternoon: linker ligation and start ligation to DNA.
Day 3 morning: test PCR, check on gel; afternoon: clean up ligation and start BstUI digest.
Day 4 morning: clean up BstUI digest; late afternoon: start HpaII digest.
Day 5 morning: PCR, freeze product, check on gel. Alternative: freeze HpaII digest.
DAY 1
STEP 1
MseI DIGESTION
1. Measure amount of isolated genomic DNA by spectrophotometry on the Nanodrop
(LGTC; 1.5 μl sample needed). Verify DNA quality by running 0.2 μg on a 0.7%
agarose gel. You should see a high-molecular smear.
2 μg genomic DNA and water
10x NEB2 buffer
10x BSA (1mg/ml)*
MseI (10U/ul) (NEB)
per sample (total 75 ul)
57 ul
7.5 ul
7.5 ul
3 ul
*Note: the BSA supplied with MseI is 100x, make a 10x working dilution!
2. Incubate in a waterbath or stove at 37˚C overnight. A heatblock generates too much
condense.
DAY 2
STEP 2
PURIFICATION OF MseI DIGEST
Heat-inactivate MseI at 65˚C for 20 minutes (heatblock) and cool to RT.
Prepare alkaline water: 1 ml milliQ water, 15 μl 0.3 M sodium bicarbonate pH 9.0.
Preheat speedvac to 60˚C (medium heat).
QIAquick purification (at RT, without pauzing):
 add 375 μl Binding Buffer PB to sample, mix and pipet onto column
 spin at 13K for 1 minute, discard flow-through
 add 750 μl Wash Buffer PE
 spin at 13K for 1 minute, discard flow-through
 dry column by spin at 13K for 1 minute
 place column in marked clean eppendorf tube
 pipet 40 μl alkaline water precisely to the middle of the column, wait 1 minute
 spin at 13K for 1 minute
 pipet 35 μl alkaline/milliQ* water precisely to the middle of the column, wait 1
minute
 spin at 13K for 1 minute
7. Remove 1.5 μl aliquot for Nanodrop DNA concentration measurement and 1 μl for gel
check.
8. Speedvac samples to less than 13.5 ul, about 20-30 minutes at 60˚C.
9. Bring volume to 13.5 μl with alkaline water.
10. Store at 4˚C until proceeding with linker ligation (end of afternoon).
*Note: It is advisable to use milliQ water in the second step to prevent accumulation
of salt in the samples. If problems with the eluation occurs switch to alkaline water.
3.
4.
5.
6.
DAY 2
STEP 3
LIGATION OF PCR LINKERS
11. Mix equal amount of both primers at RT:
per sample
3.5 ul
3.5 ul
H-12 (100 uM)
H-24 (100 uM)
12. Anneal at 55˚C to approximately RT (float tube in can with 200 ml 55˚C water from
tap or waterbath, ~2 hr).
13. Keep reaction mixture on ice (a stock of 100 μl ligated H12-H24 kept at –20˚C can be
made, prepare 7 μl aliquots).
14. Start ligation of ligated linkers to MseI digested DNA, mixing on ice in this order:
MseI digest (purified)
H-12/H-24
10x ligation buffer*
T4 DNA ligase (400 U/ul)
per sample (total 25 ul)
13.5 ul
7 ul
2.5 ul
2 ul
*Make small aliquots of 10x ligation buffer; thaw at RT (not 37˚C) to preserve rATP.
15. Ligate at 14˚C for 16 hr (960 min) >> 4˚C using PCR block (program 10).
DAY 3
STEP 4
TEST PCR
16. Keep T4-ligated DNA on ice until proceeding with purification (early afternoon).
17. Book MJ Research Dyad PCR machine for 2.5 hours (morning).
18. Make master mix for PCR on ice:
Ligated DNA
Water
DMSO
10x ThermoPol buffer (NEB)
H-24 (10 uM)
dNTP (10 mM)
DeepVent Exo- (2U/ul) (NEB)
Per sample (total 20 ul)
-1 ul13.7 ul
2 ul
2 ul
0.5 ul
0.4 ul
0.4 ul
19. Prepare PCR reactions in 200 μl thin PCR tubes or strips.
20. Use T-24-25/BOER_T25 program:
step 1: 72˚C for 5 min (extension of ligated linker to make doublestrand)
step 2: 97˚C for 1 min (denaturation)
step 3: 72˚C for 3 min (primer annealing and extension)
step 4: go to step 2 for 24 times
step 5: 72˚C for 10 min (final extension)
step 6: 18˚C for 
21. Add 5 μl 6x loading buffer to 20 μl of PCR product, and run 10 μl on 1.5% agarose gel
for 15-20 minutes (100 V for small gel). For at least one sample, run the MseIdigested DNA (Step 2). Use appropriate DNA size marker, e.g. Roch marker VI (250
ng).
22. Look for smears between 200-800 bp. If bands are not the same intensity re-adjust
concentration of DNA and restart with first step.
DAY 3
STEP 5
BstUI DIGESTION
23. Clean up ligation mixture with QIAquick PCR purification column (250 μl Binding
Buffer, elute with 40 μl and 35 μl alkaline/milliQ water).
24. Speedvac samples to 16 μl or less.
25. Store ligated DNA on ice or at 4˚C until proceeding with BstUI digestion (late
afternoon):
linker-ligated DNA, water
10x NEB2 buffer
BstUI (10 U/ul) (NEB)
per sample (total 20 ul)
16
2
2
26. Digest at 60˚C in PCR machine with heated lid for approximately 16 hours/overnight,
cool on ice.
DAY 4
STEP 6
HpaII DIGESTION
27. Clean up BstUI digest with QIAquick PCR purification column (250 μl Binding
Buffer, elute with 40 μl and 35 μl alkaline/milliQ water).
28. Speedvac samples to 16 μl or less.
29. Store BstUI-digested DNA on ice or at 4˚C until proceeding with HpaII digestion (late
afternoon):
BstUI-digested DNA, water
10x NEB1 buffer
HpaII (10 U/ul) (NEB)
per sample (total 20 ul)
16
2
2
30. Digest at 37˚C overnight in stove or waterbath, cool on ice.
DAY 5
STEP 7
PCR AMPLIFICATION OF METHYLATED DNA
31. When necessary, you can store the HpaII digest at -20˚C for later use.
32. Book MJ Research Dyad PCR machine for 2 hours (morning).
33. Set up PCR reactions:
HpaII digest
water
10x ThermoPol Buffer (NEB)
H-24 (10 uM)
dNTP (10mM each)
DeepVent Exo- (2U/ul) (NEB)
per sample (total 300 ul)
20
230.5
30
7.5
6
6
34. Make PCR master mix on ice and add 280 μl to each sample.
35. Divide the 300 μl into 2 thin wall 200 μl PCR tubes or strips.
36. Run PCR program H-24-15 (20 cycles)/BOER_L20:
step1: 72˚C for 5 min
step 2: 97˚C for 1 min
step3: 72˚C or 3 min
step 4: go to step 2 for 19 times
step 5: 72˚C for 10 min
step 6: 18˚C for 
37. Combine the content of the two parallel tubes into one mix and store on ice.
38. Assess amplification with 8 μl PCR product on 1.5% agarose gel.
39. Purify remainder (292 ul) with a QIAquick column, using 1500 μl PB buffer. For the
binding step, load the column three times with 750 ul, 750 μl and 300 μl respectively.
Discard flowthrough.
40. Vacuum dry to 40 μl or less.
41. Assess amplicon concentration by Nanodrop measurement on 1.5 ul. Expected yield
around 2 ug.
42. When yield is close to 10 μg (maximum binding capacity QIAquick column), use two
purification columns in next experiments.
43. Store the amplicons at -20˚C until ready for labeling.
Material

Enzymes: NEB/Westburg (larger sizes can be ordered as well)
o MseI (500 units) R0525S
o T4 DNA ligase (12,000 units) M0202S
o DeepVent Exo- DNA polymerase (200 units) M0259S
o BstUI (1000 units) R0518S
o HpaII (2000 units) R0171S

QIAquick PCR purification columns (Qiagen) Use fresh kit!

alkaline water: 1 ml milliQ + 15 μl 0.3 M sodium bicarbonate pH 9.0

Na-carbonate buffer: 0.3 M bicarbonbate; adjust pH to 9.0 with NaOH, store in small
aliquots at -20ºC to preserve pH.

Linkers: 40 nmole synthesis scale; e.g. Biolegio
o H-24: 5’ AGG CAA CTG TGC TAT CCG AGG GAT 3’
o H-12: 5’ TAA TCC CTC GGA 3’

10 mM each dNTP mix for PCR (250 μl):
amount stock
25 μl 100 mM dATP (Fermentas)
25 μl 100 mM dCTP (Fermentas)
25 μl 100 mM dGTP (Fermentas)
25 μl 100 mM dTTP (Fermentas)
150 μl alkaline water
final concentration
10 mM
10 mM
10 mM
10 mM
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