Noi/NOTES/2011-3-30

From ZhangLabWiki
Jump to navigation Jump to search

[[1]]

Verification of DMR.S1-S5 and DMR.Exp1-3 probe set quality and concentration by PAGE quantification[edit]

File:ZhangLab 2 2011-03-30 13hr 06min Testprobe-1.jpg File:ZhangLab 2 2011-03-30 13hr 10min Test probe2.jpg

Probe preparation[edit]

  • Dilute each probe to final concentration 2ng/ul based on original concentration
  • Mix 10ul of 2ng/ul probe with 10ul 2XTBU loading buffer
  • Mix well, heat at 75C for 8min, quick cook on ice for 3min
  • Load 6 and 3 ul in pre-run 6% TBU PAGE gel
  • Quantify concentration and calculate back to final concentration
  • Compare value of new quantification with original concentration
DMR220k Probe set Original conc. (ng/ul) New conc. (ng/ul) % different
DMR.S1 (undiluted) 26.20 30.82 14.99
DMR.S2 (undiluted) 30.10 40.38 25.46
DMR.S3 23.20 29.28 20.77
DMR.S4 24.80 26.94 7.94
DMR.S5 24.70 25.68 3.82
Exp1 33.00 31.88 3.50
Exp2 25.00 24.11 3.68
Exp3 29.00 28.90 0.35
DMR.S1 (diluted) 2.00 1.86 7.63
DMR.S2 (diluted) 2.00 1.95 2.67


Bisulfite conversion of JURKAT gDNA[edit]

Quantify bis-cvt JURKAT gDNA concentration by Qubit ssDNA assay[edit]

Sample Concentration in the Qubit ' uL used Dilution Sample Concentration ' final conc (ng/ul) Total yields in 17ul (ng)
JURKAT 1 416.00 ng/mL 1.00 200.00 416.00 ng/mL 83.20 1414.40
JURKAT 2 417.00 ng/mL 1.00 200.00 417.00 ng/mL 83.40 1417.80
JURKAT 3 365.00 ng/mL 1.00 200.00 365.00 ng/mL 73.00 1241.00
JURKAT 4 358.00 ng/mL 1.00 200.00 358.00 ng/mL 71.60 1217.20
Ave conc. 77.80 1322.60


Capture set up[edit]

Capture set Details
A MS1. DMR.S1-3 mix multiplier C
B MS2. DMR.S1-3 mix multipier D
C ME1. DMR.Exp1-3 mix
D S1 100:1 ratio
E S2 100:1 ratio
F S3 100:1 ratio
G Exp1 100:1 ratio
H Exp2 100:1 ratio
I Exp3 100:1 ratio
DMR220k Probe set Conc. (ng/ul)
DMR.S1 (undiluted) 26.20
DMR.S2 (undiluted) 30.10
DMR.S3 23.20
DMR.S4 24.80
DMR.S5 24.70
Exp1 31.88
Exp2 24.11
Exp3 28.90
DMR.S1 (diluted) 1.86
DMR.S2 (diluted) 1.95
  • Note: Still use the original concentration of some probe set since the new concentration look overestimated.

A. MS1. DMR.S1-3 mix multiplier C[edit]

  • Note: Multiplier C of S1-S3 was used for PD brain samples
Probes Amount probe required
100:1 ratio (ng)
Multiplier Amount probes required x
multiplier (ng)
Conc(ng/ul) Volume (ul) ' ' ' '
DMR220k.S1 2.23 0.05 0.11 0.10 1.12
DMR220k.S2 2.23 0.09 0.20 0.10 2.01
DMR220k.S3 18.90 0.25 4.73 2.00 2.36 B+OS mix bis-cvt JURKAT H2O Total volume
total volume 5.48 3.00 2.60 8.92 20.00


B. MS2. DMR.S1-3 mix multipier D[edit]

  • Note: Multiplier D of S1-S3 was used for Guanghui's samples (Belmonte's lab)
Probes Amount probe required
100:1 ratio (ng)
Multiplier Amount probes required x
multiplier (ng)
Conc(ng/ul) Volume (ul) ' ' ' '
DMR220k.S1 2.23 0.56 1.25 1.86 0.67
DMR220k.S2 2.23 1.00 2.23 1.95 1.14
DMR220k.S3 18.90 2.78 52.54 23.20 2.26 B+OS mix bis-cvt JURKAT H2O Total volume
total volume 4.08 3.00 2.60 10.32 20.00


C. ME1. DMR.Exp1-3 mix[edit]

Probes Amount probe required
100:1 ratio (ng)
Multiplier Amount probes required x
multiplier (ng)
Conc(ng/ul) Volume (ul) ' ' ' '
DMR220k.Exp1 18.90 1.00 18.90 31.88 0.59
DMR220k.Exp2 18.90 1.00 18.90 24.11 0.78
DMR220k.Exp3 18.90 1.00 18.90 28.90 0.65 B+OS mix bis-cvt JURKAT H2O Total volume
total volume 2.03 3.00 2.60 12.37 20.00


Group Probes Amount probe required
100:1 ratio (ng)
Multiplier Amount probes required x
multiplier (ng)
Conc(ng/ul) Volume (ul) B+OS mix bis-cvt JURKAT H2O Total volume
D S1 DMR220k.S1 2.23 1.00 2.23 1.86 1.20 3.00 2.60 13.20 20.00
E S2 DMR220k.S2 2.23 1.00 2.23 1.95 1.14 3.00 2.60 13.26 20.00
F S3 DMR220k.S3 18.90 1.00 18.90 23.20 0.81 3.00 2.60 13.59 20.00
G Exp1 DMR220k.Exp1 18.90 1.00 18.90 31.88 0.59 3.00 2.60 13.81 20.00
H Exp2 DMR220k.Exp2 18.90 1.00 18.90 24.11 0.78 3.00 2.60 13.62 20.00
I Exp3 DMR220k.Exp3 18.90 1.00 18.90 28.90 0.65 3.00 2.60 13.75 20.00


Reaction mix[edit]

Components 1rxn (ul) 11 rxn
10X AmpLigase Buffer 2.00 22.00
105nM Oligo suppressor 1.00 11.00
Total volume (ul) 3.00 aliquot 3ul to each capture tube


  • Note:
    • B = AmpLigase buffer
    • OS = Oligo suppressor

Program

   -> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h 
   -> add 2ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 100uM dNTP)    
   -> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.

Q-PCR[edit]

  • do triplicate for each sample
Components 1rxn 29x rxn mix
Template 3.00 0.00
100uM AmpF6.3 0.10 2.90
100uM AmpR6.3 0.10 2.90
Phusion HF 2x MM 25.00 725.00
50x SYBR Green 0.40 11.60
H2O 21.40 620.60
Total 50.00 aliquot 47ul, add 3ul template


Program

   98C 30sec 
   (98C 10sec -> 58C 20sec-> 72C 20sec) x 21 cycles
   72C 3min  
   15C hold.

Q-PCR Result[edit]

Probe set Ct Mean Ct Dev. Ratio compared to s1 Probes size Adjusted Ratio % of s1
S1-3 MultiC 17.83 0.34 1.17 68000 0.11 11.21%
S1-3 MultiD 15.54 0.14 5.74 68000 0.55 54.83%
Exp1-3mix 15.10 0.27 7.78 165000 0.31 30.65%
S1 100:1 18.06 0.19 1.00 6500 1.00 100.00%
S2 100:1 18.60 0.27 0.69 6500 0.69 68.78%
S3 100:1 16.66 0.20 2.64 55000 0.31 31.19%
Exp1 100:1 17.21 0.31 1.80 55000 0.21 21.30%
Exp2 100:1 16.87 0.02 2.28 55000 0.27 26.96%
Exp3 100:1 17.39 0.07 1.59 55000 0.19 18.80%


PAGE analysis[edit]

File:ZhangLab 2 2011-04-06 13hr 58min PAGE Q-PCR2ul.jpg

  • Loaded sample 2ul
  • Repeated Q-PCR again using the same condition by running for 18 cycles to be able to compare the band intensity of each probe set

PAGE analysis[edit]

File:ZhangLab 2 2011-04-06 17hr 16min PAGE Q-PCR 1-5ul.jpg

  • Loaded sample 1.5ul

Result from PAGE quantification[edit]

Probe set Ratio compared to s1 Adjusted Ratio % of s1
S1-3 MultiC 0.97 0.09 9.26%
S1-3 MultiD 4.30 0.41 41.12%
Exp1-3mix 5.31 0.21 20.90%
S1 100:1 1.00 1.00 100.00%
S2 100:1 0.62 0.62 61.98%
S3 100:1 1.75 0.21 20.67%
Exp1 100:1 1.52 0.18 17.96%
Exp2 100:1 2.17 0.26 25.62%
Exp3 100:1 1.77 0.21 20.97%


  • A = S1-3 MultiC
  • B = S1-3 MultiD
  • C = Exp1-3 mix
  • D = S1 100:1
  • E = S2 100:2
  • F = S3 100:3
  • G = Exp1 100:1
  • H = Exp2 100:1
  • I = Exp3 100:1
  • From this result, PAGE analysis supports the Q-PCR result