AlanFung:LabNotes/Capturing/Ms Meth/EoH;E4H;E24H/2009-6-9

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Material[edit]

  • Ampligase DNA Ligase 1000U @ 5U/uL Epicentre Cat. No. A8101
  • dNTP Mix-Deoxynucleotide Solution Mix 10nM NEB #N0447L
  • AmpliTaq DNA Polymerase Stoffel Fragment 1000U @ 10U/uL Applied Biosystem Part# N808-0038
*Mouse Samples
*EoH 45ng/uL*50uL
*E4H 45ng/uL*50uL
*E24H 50ng/uL*42uL

Objective[edit]

  • Bisulfite treatment of all 3 mouse samples
  • Capture targeted sequence with Ms_Meth probe sets
  • PCR amplification of the captured sequence
  • TBE gel verification of the captured sequence

Overview[edit]

  • Bisulfite Conversion of DNA
  • Capturing Reaction

Reagent Preparation[edit]

 dNTP concentration 10mM(NEB)
                                                                    ddH2O     Ligase Buffer   Ligase     Amplitaq    dNTP           
 2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 1mM dNTP:      0.5ul         0.1ul       0.1ul       0.2ul    (10mM)0.1ul 
                                                           x30       15ul          3ul        3ul        6ul      3ul


Procedures[edit]

Bilsulfite Conversion with EZ DNA Methylation-Gold Kit[edit]

*EoH 45ng/uL*50uL
*E4H 45ng/uL*50uL
*E24H 50ng/uL*42uL
  • Samples are too dilute to get 2ug(max for the methylation column) from each sample for conversion
  • Take 20uL from each tube to perform CT conversion
  • Add 900ul ddH2O, 300ul M-Dilution Buffer and 50ul M-Dissolving Buffer to a tube of CT Conversion Reagent vortex for 10M
  • Add 130ul of CT conversion reagent to each 20uL sample
  • Mix the sample and perform
  • 98C 10min -> 64C 150min -> 4c overnight.
  • Add 600ul M-Binding buffer to spin columns.
  • Add the converted samples to the columns, close the gap and mix by inverting several times.
  • Spin at 15,000rpm for 30sec, discard flow through
  • Add 100ul M-Wash Buffer, spin for 30 sec, discard flow-through.
  • Add 200ul M-Desulphonation Buffer, wait for 20min, spin at 15,000 rpm for 30sec.
  • Add 200ul M-Wash Buffer, spin for 30 sec.
  • Place the columns into 1.5ml tubes, add 10ul M-Elution Buffer. Wait for 1min, spin at 15,000rpm for 30sec.
  • Measure the DNA with Nanodrop:
*EoH (CT Converted) 81.8ng/uL * 8.5uL
*E4H (CT Converted) 94.1ng/uL * 8.5uL
*E24H (CT Converted) 95.4ng/uL *8.5uL


Calculation for Ms_Meth Probe Set[edit]

Probe:target ratio 200:1 
EoH CT Converted Template(81.8ng/uL)
E4H CT Converted Template(94.1ng/uL)
E24H CT Converted Template(95.4ng/uL)

To get 300ng
EoH needs 3.667uL
E4H needs 3.188uL
E24H needs 3.145uL
  • One genome mass = 3pg
  • First calculate the # of genome your target represents
  • For 200ng, it represents 200ng/3pg=66666.67 genomes
  • You want to have 200x the probes as the target 200ng/3pg*200
  • You want every single probe to target ratio to be 200:1 200ng/3pg*200*55,000
Ms_Meth 22ng/uL 55,000Probes 100nt
Since 1 base = 330Da (for single stranded DNA, 660Da for ds), 
the concentration of probe is 22 ng/ul /(100x330) = 0.000666667 = 666.67nM
# of molecules per mole = 6X10^23
# of probes needed/ concentration of probes = Volume of probes needed
the volume the probe needed is 200ng/3pg x 200 x 55,000 / [(6x10^23)x (666.67*10^-9M)]*10^6 =1.83uL

Capturing System Setup[edit]

                                       EoH      E4H      E24H      +ve       -ve
  300ng CT Converted Samples           3.667uL  3.188uL  3.145uL   1.5uL     0uL  
  10X AmpLigase buffer                 1uL      1uL      1uL       1uL       1uL
  Ms_Meth Probe (666.67nM)             1.83uL   1.83uL   1.83uL    1.83uL    1.83uL      
  H2O                                  3.503uL  3.982uL  4.025uL   5.67uL    7.17uL
  Total                                10uL     10uL     10uL      10uL      10uL
95c 10min -> 60C 24h ->add 2ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 1mM dNTP) -> 60C 4h -> 
4 cycels (95C 1min -> 55C for 4h),add 2ul more SLN mix after 1st cycle -> 95C 5min ->
add 2ul Exonuclease I/III mixadd 2ul Exonuclease I/III mix as soon as the temperature is lowered to 37C -> 37C 1h -> 94C 5min -> 4C hold

PCR with AmpF/R6.2Sol and iProof[edit]

                           EoH     E4H      E24H    +ve    -ve
  DNA Template             5uL     5uL      5uL     5uL    5uL
  2X iProof Mastermix      50uL    50uL     50uL    50uL   50uL
  AmpF6.3 (10uM)           4uL     4uL      4uL     4uL    4uL
  AmpR6.3 (10uM)           4uL     4uL      4uL     4uL    4uL
  50X SYBG I               0.4uL   0.4uL    0.4uL   0.4uL  0.4uL
  H2O                      36.6uL  36.6uL   36.6uL  36.6uL 36.6uL

98C 30S -> (98C 10S -> 58C 20S -> 72C 20S->plate read) x 8 -> (98C 10S -> 72C 20S->plate read) x 8-> 72C 5 min -> 15C hold.

  • Signal not increasing
  • Stopped at 8th cycle, replace 2nd cycle with first one, for AmpF/R6.3 don't have long tail as AmpF/R6.3Sol hence we dont use high temp. annealing

TBE Gel Verification[edit]

  • 0.5uL low mass ladder + 3uL 6X gel loading dye
  • 3uL amplified EoH + 3uL 6X gel loading dye
  • 3uL amplified E4H + 3uL 6X gel loading dye
  • 3uL amplified E24H + 3uL 6X gel loading dye
  • 3uL amplified +ve + 3uL 6X gel loading dye
  • 3uL amplified -ve + 3uL 6X gel loading dye

Purification with Qiaquick column[edit]

  • Add 5X sample volume of PB buffer into each tube
  • Mix by repeat pipetting
  • Load mixture onto column
  • Centrifuge at 14,000rpm for 1min
  • Discard flow through and add 750uL PE buffer to each column
  • Repeat Centrifuge
  • Discard flow through
  • Repeat centrifuge
  • Transfer columns to clean 1.5mL tubes
  • Place the column/tube assemblies on bench top wait for 2min
  • Add in 50uL EB Buffer to each column (For nicking enzyme probes add in 100ul of ddH2O) let stand for 5min
  • Repeat centrifuge

E-gel size selection2%[edit]

1        2         3         4        M        5        6         7          8

30uL-EB  15uL+ve   30uL-EoH  30uL-EB  1.5uL-L  30uL-EB  30uL-E4H  30uL-E24H  30uL-EB
         15uL-EB                      10uL-EB 
30uL-EB  30uL-EB   30uL-EB   30uL-EB  30uL-EB  30uL-EB  30uL-EB   30uL-EB    30uL-EB
  • Run Mode3 (Run E-Gel) -DC ~15M
  • Collect samples ~400bp w/ P200 pipette
  • Add in 20ul EB, continue run
  • Run Mode 5 (Reverse)
  • Repeat run and reverse a couple of times meanwhile collected the samples

TBE Gel quantification[edit]

.5uL low mass ladder + 3.5uLddh2o + 3uL 6X gel loading dye (6uL to well)
3uL EoH sample + 3ul 6x gel loading dye (6uL to well)
3ul E4H sample + 3ul 6x gel loading dye (6uL to well)
3uL E24H sample + 3ul 6x gel loading dye (6uL to well)
3ul +ve control + 3ul 6x gel loading dye (6uL to well)
3ul 90-mer(5ng/ul) + 3ul 6x gel loading dye (4uL + 2uL to well)

File:ZhangLab 2 2009-06-12 10hr 51min.jpg

Egel Size Selection[edit]

  • Prepare tube with 750uL EB buffer
  • Label clean tubes for samples
  • Use only well #2,3,6,7
  • Use M for Ladder
  • Fill any unused wells w/ 30ul EB buffer
  • Load 1.5ul low mass ladder + 10ul EB buffer to well M
  • Load 30ul samples to well 3,6 & 7
  • Load 15ul +ve control + 15ul EB buffer to well 2
  • Run <Mode 3> <Run E-Gel-DC> ~15M
  • Pause when samples of interest reach the bottom well
  • Aspirate samples w/ P200 pipette to the clean labeled tube
  • Re-fill bottom well with 15ul EB buffer and continue run and aspirate procedure until all samples of interest are withdrawn
  • Run TBE gel for quantification

File:ZhangLab 2 2009-06-12 17hr 34min.jpg

E24H 6.56 ng/ul * 46uL (26nM)
E4H 19.6 ng/ul * 40uL	(40nM)
EoH 43.53 ng/ul * 75uL (88nM)

20090615 to Yuan Gao: refer to LabNotes on [1]

 Hybrid_1_CES27k9bp: 26nM x 15ul
 BJ_CES27k9bp: 39nM x 15ul
 The above 2 samples for end-sequencing.
 the following 3 samples are captured targets of mouse DNA.refer to LabNotes on [2]
 E24H 6.56 ng/ul * 46uL (26nM)
 E4H 19.6 ng/ul * 40uL	(40nM)
 EoH 43.53 ng/ul * 75uL (88nM)