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
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 |
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
- 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.