AlanFung:LabNotes/Capturing/CPG97K Low-input/2009-10-24

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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

Overview[edit]

  • Bisulfite Conversion of DNA
  • Capturing Reaction

Calculation[edit]

Probe:target ratio 200:1 (according to Alice, 100:1 works too)
*Billy uses 250ng
  • CT converted Jurkat gDNA (200ng/ul)
  • CT converted GM20431 (11.6ng/ul)
  • According to Ida low-input works on 50ng, test with 100ng first
  • to get 100ng
  • Need- 0.5ul Jurkat gDNA
  • 8.62ul GM20431 gDNA


  • 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

Procedures[edit]

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

  • Add 900ul ddH2O, 300ul M-Dilution Buffer and 50ul M-Dissolving Buffer to a tube of CT Conversion Reagent vortex for 10M
  • Add 24mL of 100% ethanol to the 6mL M-Wash Buffer concentrate
  • Add 130ul of CT conversion reagent to samples. If your DNA sample is less than 20uL make up the difference with H2O
  • 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 Qubit ssDNA kit:


  • CT Converted GM20431 gDNA concentration: 11.6ng/ul
  • Concentration of CT converted Jurkat gDNA: 200ng/ul
  • Concentration of N6 hexamer: 10ng/ul

Capturing System Setup[edit]

  • Prepare total volume up to 15ul
50ng input 30ng input
Capturing System Jurkat GM20431 Negative Control Jurkat GM20431 Negative Control
Volume of N6 (10ng/ul) 5.000 5.000 0.000 7.000 7.000 0.000
Volume of probes (0.7ng/ul) 3.929 3.929 3.929 2.357 2.357 2.357
Volume of templates 2.500 4.310 0.000 1.500 2.586 0.000
Volume of H20 2.071 0.261 9.571 2.643 1.557 11.143
Volume of 10X Ampligase buffer 1.500 1.500 1.500 1.500 1.500 1.500
Total Volume 15.000 15.000 15.000 15.000 15.000 15.000
10ng input 6ng input
Capturing System Jurkat GM20431 Negative Control Jurkat GM20431 Negative Control
Volume of N6 (10ng/ul) 9.000 9.000 0.000 9.400 9.400 0.000
Volume of probes 0.786 0.786 0.786 0.471 0.471 0.471
Volume of templates 0.500 0.862 0.000 0.300 0.517 0.000
Volume of H20 3.214 2.852 12.714 3.329 3.111 13.029
Volume of 10X Ampligase buffer 1.500 1.500 1.500 1.500 1.500 1.500
Total Volume 15.000 15.000 15.000 15.000 15.000 15.000


 95c 10min -> 55C 16h ->add 1ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.2mM dNTP) -> 55C 4h -> 
9 cycels (95C 1min -> 55C for 4h) -> 95C 1min -> 37C 1min ->
add 2ul Exonuclease I/III mix -> 37C 2h -> 94C 5min -> 4C hold.

PCR(01/26/2009)[edit]

  • Scale down template to 2ul and run till saturation, to observe the result of different amount of template input
1well (ul) X15 /5
Template 10 150 30
2X Phusion 50 750 150
AmpF6.2SoL (10uM) 4 60 12
AmpR6.2Sol (10uM) 4 60 12
50X SYBG I 0.8 12 2.4
H2O 31.2 468 93.6
  • Plate Setup
Setup
50JT 10G
50J 10N
50G 6J
50N 6G
30J 6N
30G
30N
10J

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

  • Update after 11/09/09 meeting (Need to move on to the next step on the middle panel)
  • New qPCR result

File:11 10 09 ALL.jpg

11/6/09[edit]

11/7/09[edit]

  • Repeat amplification using all of the 50ng captured product
1well (ul) X3 X1.1
Template 10
2X Phusion 50 150 165
AmpF6.2SoL (10uM) 4 12 13.2
AmpR6.2Sol (10uM) 4 12 13.2
50X SYBG I 0.8 2.4 2.64
H2O 31.2 93.6 102.96

File:11 7 09 QPCR.jpg

11/10/09[edit]

  • Run a TBE Gel for all amplified products
  • Gel Setup (2 10well gels)
L|50J|50G|50N||30J|30G|30N||
L|10J|10G|10N||6J|6G|6N||
Template 6X Laoding Dye H2O
J 5 5 5
G 5 5 5
N 5 5 5
  • gel stain issue, very strong band covering everything, repeat with new setup

11/11/09[edit]

  • Repeat qPCR, stop when samples reached plateau

File:20091110 172133.jpg

  • Run a TBE gel with 1KB ladder

File:ZhangLab 2 2009-11-11 16hr 57min.jpg

  • something wrong with the 1KB ladder, huge smear indicates over amplification
  • Repeat qPCR stop 3 cycles before it ends

11/12/09[edit]

1well (ul) X 8 X1.1
Template 10
2X Phusion 50 400 440
AmpF6.2SoL (10uM) 4 32 35.2
AmpR6.2Sol (10uM) 4 32 35.2
50X SYBG I 0.8 6.4 7.04
H2O 31.2 249.6 274.56

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