Noi/NOTES/2011-2-15: Difference between revisions

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**Gr. B = P4C
**Gr. B = P4C
**Gr. C = P8P2
**Gr. C = P8P2
* From PGP amplification, high percentage of N2Ligated DN template didn't show a good yield. I will reduce the amount of template to 1%, 2% and 5% and also reduce amount of primers to 1ul of 10uM in total volume 100ul (this exp do 50ul then just in proportion).
* Note: this volume based on 100ng of MmeI digested DNA ligated to N2 adapters and purify with Ampure beads eluted volume 30ul.
{| {{table}} border = 1
{| {{table}} border = 1
| align="center" style="background:#f0f0f0;"|'''Reagents'''
| align="center" style="background:#f0f0f0;"|'''Reagents'''
| align="center" style="background:#f0f0f0;"|'''1% template'''
| align="center" style="background:#f0f0f0;"|'''4.2rxn mix'''
| align="center" style="background:#f0f0f0;"|'''2% template'''
| align="center" style="background:#f0f0f0;"|'''2% template'''
| align="center" style="background:#f0f0f0;"|'''4.2rxn mix'''
| align="center" style="background:#f0f0f0;"|'''4.2rxn mix'''
| align="center" style="background:#f0f0f0;"|'''5% template'''
| align="center" style="background:#f0f0f0;"|'''5% template'''
| align="center" style="background:#f0f0f0;"|'''4.2rxn mix'''
| align="center" style="background:#f0f0f0;"|'''10% template'''
| align="center" style="background:#f0f0f0;"|'''4.2rxn mix'''
| align="center" style="background:#f0f0f0;"|'''4.2rxn mix'''
|-
|-
| N2 adapter ligated template||1.00||0.00||2.50||0.00||5.00||0.00
| N2 adapter ligated template||0.50||0.00||1.00||0.00||2.50||0.00
|-
|-
| 10uM PCR_F||1.00||4.20||1.00||4.20||1.00||4.20
| 10uM PCR_F||0.50||2.10||0.50||2.10||0.50||2.10
|-
|-
| 10uM PCR_R.N2 IndX(X= 1, 2 and 4)||1.00||0.00||1.00||0.00||1.00||0.00
| 10uM PCR_R.N2 IndX(X= 1, 2 and 4)||0.50||0.00||0.50||0.00||0.50||0.00
|-
|-
| 50x SYBG||0.20||0.84||0.20||0.84||0.20||0.84
| 50x SYBG||0.20||0.84||0.20||0.84||0.20||0.84
Line 515: Line 517:
| 2x PhusionHF MM||25.00||105.00||25.00||105.00||25.00||105.00
| 2x PhusionHF MM||25.00||105.00||25.00||105.00||25.00||105.00
|-
|-
| H2O||21.80||91.56||20.30||85.26||17.80||74.76
| H2O||23.30||97.86||22.80||95.76||21.30||89.46
|-
|-
| Total volume (ul)||50.00||aliquot 48ul||50.00||aliquot 46.5ul||50.00||aliquot 44ul
| Total volume (ul)||50.00||aliquot 49ul||50.00||aliquot 48.5ul||50.00||aliquot 47ul
|-
|-
| ||||add 1ul template||||add 2.5 template||||add 5ul template
| |||| add 0.5ul template|||| add 1ul template||||add 2.5ul template
|-
|-
| ||||add 1ul R primer||||add 1ul R primer||||add 1ul R primer
| ||||add 0.5ul R primer||||add 0.5ul R primer||||add 0.5ul R primer
|}<br>
|}<br>



Revision as of 02:56, 25 February 2011

DMR220k capture(Probe set S1-S5 plus new probe set Exp1-Exp3) and library construction of Parkinson's patients

  • Received 18 human brain samples from Pakinson's patients. This includes control and Pakinson's patiants (2011_02_02)
  • Human frozen cerebral cortex
Pair Control Sample ID Sample code Sex age (year.day) Postmortem time Conc. (ug/ul)/ volume (ul) PD Sample ID Sample code Sex
1. 5028 P1C M 67.29 P18 0.5ug/ul/ 4ul 1947 P1P M
2. 4789 P2C F 72.05 P19 0.5ug/ul/ 4ul 4977 P2P F
3. 5171 P3C M 79.09 P05 0.5ug/ul/ 4ul 4879 P3P M
4. 1818 P4C M 76.29 P03 0.5ug/ul/ 4ul 4526 P4P M
5. 5089 P5C M 89.02 P14 0.5ug/ul/ 4ul 5203 P5P M
6. 5237 P6C M 52.29 P13 0.5ug/ul/ 4ul 1910 P6P M
7. 1569 P7C1 F 77.09 P08 0.5ug/ul/ 4ul 1401 P7P F
7. 5219 P7C2 F 76.35 P03 0.5ug/ul/ 4ul
8. 4735 P8C M 73.18 P21 0.5ug/ul/ 4ul 1741 P8P1 M
8. 5306 P8P2 M


Bisulfite conversion

  • gDNA total volume 4ul (0.5ng/ul) so total amount = 2ug
  • Add EB 6 ul to make volume to 10ul, so the final concentration should be 200ng/ul
Sample ID Conc.(ng/ul) Volume (ul) H20 (ul) CT conversion reagent (ul) Total volume (ul)
P1C 200.00 5.00 15.00 130.00 150.00
P2C 200.00 5.00 15.00 130.00 150.00
P3C 200.00 5.00 15.00 130.00 150.00
P4C 200.00 5.00 15.00 130.00 150.00
P5C 200.00 5.00 15.00 130.00 150.00
P6C 200.00 5.00 15.00 130.00 150.00
P7C1 200.00 5.00 15.00 130.00 150.00
P7C2 200.00 5.00 15.00 130.00 150.00
P8C 200.00 5.00 15.00 130.00 150.00
P1P 200.00 5.00 15.00 130.00 150.00
P2P 200.00 5.00 15.00 130.00 150.00
P3P 200.00 5.00 15.00 130.00 150.00
P4P 200.00 5.00 15.00 130.00 150.00
P5P 200.00 5.00 15.00 130.00 150.00
P6P 200.00 5.00 15.00 130.00 150.00
P7P 200.00 5.00 15.00 130.00 150.00
P8P1 200.00 5.00 15.00 130.00 150.00
P8P2 200.00 5.00 15.00 130.00 150.00


  • follow standard protocol for bisulfite conversion of ZymoResearch
  • elute with 17ul Elution buffer

Determine bisulfite converted gDNA concentration using Qubit Fluorometer

Sample preparation

  • Add 1ul of BIS-CVT gDNA to 199ul of Quant-iT working solution
Sample ID Concentration in the Qubit ' uL used Dilution Sample Concentration ' Final conc.(ng/ul) Yields (ng)
P1C 150.00 ng/mL 1.00 200.00 150.00 ng/mL 30.00 510.00
P2C 118.00 ng/mL 1.00 200.00 118.00 ng/mL 23.60 401.20
P3C 59.40 ng/mL 1.00 200.00 59.40 ng/mL 11.88 201.96
P4C 321.00 ng/mL 1.00 200.00 321.00 ng/mL 64.20 1091.40
P5C 34.70 ng/mL 1.00 200.00 34.70 ng/mL 6.94 117.98
P6C 29.10 ng/mL 1.00 200.00 29.10 ng/mL 5.82 98.94
P7C1 33.70 ng/mL 1.00 200.00 33.70 ng/mL 6.74 114.58
P7C2 29.60 ng/mL 1.00 200.00 29.60 ng/mL 5.92 100.64
P8C 57.60 ng/mL 1.00 200.00 57.60 ng/mL 11.52 195.84
P1P 189.00 ng/mL 1.00 200.00 189.00 ng/mL 37.80 642.60
P2P 393.00 ng/mL 1.00 200.00 393.00 ng/mL 78.60 1336.20
P3P 337.00 ng/mL 1.00 200.00 337.00 ng/mL 67.40 1145.80
P4P 307.00 ng/mL 1.00 200.00 307.00 ng/mL 61.40 1043.80
P5P 19.10 ng/mL 1.00 200.00 19.10 ng/mL 3.82 64.94
P6P 48.00 ng/mL 1.00 200.00 48.00 ng/mL 9.60 163.20
P7P 18.90 ng/mL 1.00 200.00 18.90 ng/mL 3.78 64.26
P8P1 44.60 ng/mL 1.00 200.00 44.60 ng/mL 8.92 151.64
P8P2 37.80 ng/mL 1.00 200.00 37.80 ng/mL 7.56 128.52


Capture set up

Probe calculation

Probe/target ratio 100 '
Probe size (sub set Exp1-3) 55,000 each
Template 200ng
Human gDNA MW 1.82E+12g/mol (3E9bp x 607.4D/bp + 157)
Human gDNA 200ng 1.10E-19mol
Probe required (100:1) 1.10E-17 mol
Probe MW (each set) 1.72E9 g/mol (55,000 x 103bp x 303.7D/bp + 79D)
Amount probe required 18.90ng each
  • Note: for DMR220k subset S1-S5 used Multiplier C
Subset ID Multiplier C Amount probes required x multiplier (ng) Conc(ng/ul) Volume (ul) 20 rxn
DMR.s1 0.05 0.11 2.00 0.06 1.10
DMR.s2 0.09 0.21 2.00 0.11 2.10
DMR.s3 0.25 4.71 23.20 0.20 4.06
DMR.s4 1.00 18.88 24.80 0.76 15.23
DMR.s5 4.83 91.22 24.70 3.69 73.86
DMR.Exp1 1.00 18.90 28.00 0.68 13.50
DMR.Exp2 1.00 18.90 26.00 0.73 14.54
DMR.Exp3 1.00 18.90 25.00 0.76 15.12
Total volume (ul) 6.98 139.51
  • Note: used probe set DMR220k S5, Exp1-3 prepared on 2011_02_02

Reaction mix

Components 1rxn (ul) 20rxn mix (ul)
10X AmpLigase Buffer 2.55 51.00
DMR220k Exp1-3 probe mix (ul) 6.98 139.60
105nM Oligo suppressor 1.00 20.00
Total volume (ul) 10.53 210.60


Sample ID Final conc.(ng/ul) volume for 200ng H2O probe mix (ul) 10x AmpLigase buffer (ul) 105nM Oligo suppressor (ul) Total volume (ul) Total amount of bis-cvt DNA captured
P1C 30.00 6.67 8.33 6.98 2.55 1.00 25.53 200.00
P2C 23.60 8.47 6.53 6.98 2.55 1.00 25.53 200.00
P3C* 11.88 15.00 0.00 6.98 2.55 1.00 25.53 178.20
P4C 64.20 3.12 11.88 6.98 2.55 1.00 25.53 200.00
P5C* 6.94 15.00 0.00 6.98 2.55 1.00 25.53 104.10
P6C* 5.82 15.00 0.00 6.98 2.55 1.00 25.53 87.30
P7C1* 6.74 15.00 0.00 6.98 2.55 1.00 25.53 101.10
P7C2* 5.92 15.00 0.00 6.98 2.55 1.00 25.53 88.80
P8C* 11.52 15.00 0.00 6.98 2.55 1.00 25.53 172.80
P1P 37.80 5.29 9.71 6.98 2.55 1.00 25.53 200.00
P2P 78.60 2.54 12.46 6.98 2.55 1.00 25.53 200.00
P3P 67.40 2.97 12.03 6.98 2.55 1.00 25.53 200.00
P4P 61.40 3.26 11.74 6.98 2.55 1.00 25.53 200.00
P5P* 3.82 15.00 0.00 6.98 2.55 1.00 25.53 57.30
P6P* 9.60 15.00 0.00 6.98 2.55 1.00 25.53 144.00
P7P* 3.78 15.00 0.00 6.98 2.55 1.00 25.53 56.70
P8P1* 8.92 15.00 0.00 6.98 2.55 1.00 25.53 133.80
P8P2* 7.56 15.00 0.00 6.98 2.55 1.00 25.53 113.40
  • Note: * less than 200ng of bis-cvt DNA in probe capture

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; 50uM dNTP)  (2011_02_16)      
   -> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.

PCR Amplification

(2011_02_17)

  • amplify 2tubes/ sample
  • 19samples x 2 = 38 plus 1 NTC = 39rxn
Reagents volume (ul) 39.5rxn mix
captured template 9.00 0.00
100uM AmpFV6.3NH2 0.20 7.90
100uM AmpRV6.3NH2 0.20 7.90
50x SYBG 0.40 15.80
2x PhusionHF MM 50.00 1975.00
H2O 40.20 1587.90
Total volume (ul) 100.00 3594.50

aliquot 91ul, add 9ul of captured DNA template

Program

   98C 30sec 
   (98C 10sec -> 58C 20sec-> 72C 20sec) x 20 cycles
   72C 2min  
   15C hold.
- purify with 0.7vol. Ampure beads, elute with 25ul EB/tube, so total volume = 75ul
- PAGE quantify DNA concentration (loaded sample 1ul)

File:ZhangLab 2 2011-02-22 15hr 46min 1st Amp P1C-P8C.jpg File:ZhangLab 2 2011-02-22 15hr 46min 1st Amp P1P-P2P2.jpg

PAGE quantification and MmeI digestion set up

Sample ID Conc.(ng/ul) Total volume(ul) Total amount (ng) 10X NEB buffer4 32mM SAM MmeI Total volume(ul)
P1C 2.09 75.00 156.39 9.30 0.30 8.00 92.60
P2C 4.67 75.00 350.41 9.30 0.30 8.00 92.60
P3C 3.09 75.00 231.84 9.30 0.30 8.00 92.60
P4C 4.24 75.00 317.74 9.30 0.30 8.00 92.60
P5C 3.45 75.00 258.94 9.30 0.30 8.00 92.60
P6C 4.19 75.00 314.13 9.30 0.30 8.00 92.60
P7C1 3.74 75.00 280.28 9.30 0.30 8.00 92.60
P7C2 2.14 75.00 160.76 9.30 0.30 8.00 92.60
P8C 3.21 75.00 240.62 9.30 0.30 8.00 92.60
P1P 3.35 75.00 251.00 9.30 0.30 8.00 92.60
P2P 3.18 75.00 238.35 9.30 0.30 8.00 92.60
P3P 3.01 75.00 225.81 9.30 0.30 8.00 92.60
P4P 5.10 75.00 382.61 9.30 0.30 8.00 92.60
P5P 4.06 75.00 304.36 9.30 0.30 8.00 92.60
P6P 3.22 75.00 241.75 9.30 0.30 8.00 92.60
P7P 3.68 75.00 276.35 9.30 0.30 8.00 92.60
P8P1 4.03 75.00 302.36 9.30 0.30 8.00 92.60
P8P2 7.34 75.00 550.50 9.30 0.30 8.00 92.60


Reagents 1x 18.5 rxn mix
10X NEB buffer4 9.30 172.05
32mM SAM 0.30 5.55
MmeI 8.00 148.00
Total 17.60 aliquot 17.6ul, add 75ul of amplified amplicon


  • incubate 37C 2hr
  • purify with 0.7vol. Ampure beads, elute with 30ul EB
  • PAGE quantify DNA concentration (loaded sample 2ul)

File:ZhangLab 2 2011-02-22 20hr 38min MmeI digestion P1C-P8C.jpg File:ZhangLab 2 2011-02-22 20hr 41min MmeI digestion P1P-P8P2.jpg

Sample ID Conc of MmeI completely digested DNA (ng/ul) Total volume (ul) Total amount (ng)
P1C 0.56 30.00 16.84
P2C 1.60 30.00 47.94
P3C 0.97 30.00 28.97
P4C 1.33 30.00 39.91
P5C 1.42 30.00 42.46
P6C 0.99 30.00 29.61
P7C1 1.41 30.00 42.35
P7C2 0.46 30.00 13.71
P8C 0.57 30.00 17.01
P1P 0.59 30.00 17.62
P2P 0.94 30.00 28.29
P3P 0.61 30.00 18.17
P4P 1.59 30.00 47.80
P5P 0.92 30.00 27.58
P6P 0.42 30.00 12.64
P7P 1.15 30.00 34.45
P8P1 1.29 30.00 38.72
P8P2 2.03 30.00 60.95


N2 adaptor ligation set up

  • group sample into 3 different groups based on total completely MmeI digested DNA amount including
    • groupA: <= 20ng
    • 20ng < groupB <= 40ng
    • 40ng < groupC <= 60ng

Adaptor preparation

Components Volume (ul) Final conc. (uM)
100uM PE_N2_adaptor 2.00 20.00
100uM PE_b_A 2.00 20.00
10x Stoffel buffer 1.00 1x
H2O 5.00
Total 10.00


Program

94C 2min -> 0.2C/sec to 20C -> 4C hold

Adapters to ligated product ratio: 10:1
Est length of digested products: 212bp (after MmeI)
MW digested products = (212bp*607.4 D/bp +157.9 D) = 128.927kD = 128,927 g/mole

For 100 ng digested product = 100ng / 128,927g/mole = 7.76E-4 nmole * 10:1 = 0.008 nmole adapters required.
20uM Adapters (ul) = 0.008nmoles/ (20xE3nmoles/L * 1E-6L/ul) = 0.008nmoles/ (20E-3 nmoles/ul) = 0.40 ul

  • 0.40 ul of 20uM adapters per 100ng digested products.
  • so for lower amount of MmeI digested DNA, dilute adaptor with different dilution
Group (by DNA amount) Samples conc.(ng/ul) Volume (ul) Total amount (ng) N2 adapters conc (uM) N2 adaptors volume (ul) 5xQuickLigase buffer QuickLigase (ul) H2O (ul) Total (ul)
A P1C 0.56 30.00 16.84 4.00 0.40 8.00 1.00 0.60 39.40
C P2C 1.60 30.00 47.94 12.00 0.40 8.00 1.00 0.60 39.40
B P3C 0.97 30.00 28.97 8.00 0.40 8.00 1.00 0.60 39.40
B P4C 1.33 30.00 39.91 8.00 0.40 8.00 1.00 0.60 39.40
B P5C 1.42 30.00 42.46 8.00 0.40 8.00 1.00 0.60 39.40
B P6C 0.99 30.00 29.61 8.00 0.40 8.00 1.00 0.60 39.40
B P7C1 1.41 30.00 42.35 8.00 0.40 8.00 1.00 0.60 39.40
A P7C2 0.46 30.00 13.71 4.00 0.40 8.00 1.00 0.60 39.40
A P8C 0.57 30.00 17.01 4.00 0.40 8.00 1.00 0.60 39.40
A P1P 0.59 30.00 17.62 4.00 0.40 8.00 1.00 0.60 39.40
B P2P 0.94 30.00 28.29 8.00 0.40 8.00 1.00 0.60 39.40
A P3P 0.61 30.00 18.17 4.00 0.40 8.00 1.00 0.60 39.40
C P4P 1.59 30.00 47.80 12.00 0.40 8.00 1.00 0.60 39.40
B P5P 0.92 30.00 27.58 8.00 0.40 8.00 1.00 0.60 39.40
A P6P 0.42 30.00 12.64 4.00 0.40 8.00 1.00 0.60 39.40
B P7P 1.15 30.00 34.45 8.00 0.40 8.00 1.00 0.60 39.40
B P8P1 1.29 30.00 38.72 8.00 0.40 8.00 1.00 0.60 39.40
C P8P2 2.03 30.00 60.95 12.00 0.40 8.00 1.00 0.60 39.40


Reagents 1x Mix A (6.5 rxn mix) Mix B (9.5 rxn mix) Mix C (4.5 rxn mix)
5xQuickLigase buffer 8.00 52.00 76.00 36.00
QuickLigase 1.00 6.50 9.50 4.50
N2 Adapters, conc. (Gr. A, B or C) 0.40 2.60 3.80 1.80
H2O 0.60 3.90 5.70 2.70
Total 10.00 65.00 95.00 45.00


- incubate at RT for 15 min
- purify with Ampure beads, elute with 30ul EB

Amplification with N2 barcoded primers

  • vary volume of DNA template 2%, 5% and 10% in different group A, B and C
    • Gr. A = P1C
    • Gr. B = P4C
    • Gr. C = P8P2
  • From PGP amplification, high percentage of N2Ligated DN template didn't show a good yield. I will reduce the amount of template to 1%, 2% and 5% and also reduce amount of primers to 1ul of 10uM in total volume 100ul (this exp do 50ul then just in proportion).
  • Note: this volume based on 100ng of MmeI digested DNA ligated to N2 adapters and purify with Ampure beads eluted volume 30ul.
Reagents 1% template 4.2rxn mix 2% template 4.2rxn mix 5% template 4.2rxn mix
N2 adapter ligated template 0.50 0.00 1.00 0.00 2.50 0.00
10uM PCR_F 0.50 2.10 0.50 2.10 0.50 2.10
10uM PCR_R.N2 IndX(X= 1, 2 and 4) 0.50 0.00 0.50 0.00 0.50 0.00
50x SYBG 0.20 0.84 0.20 0.84 0.20 0.84
2x PhusionHF MM 25.00 105.00 25.00 105.00 25.00 105.00
H2O 23.30 97.86 22.80 95.76 21.30 89.46
Total volume (ul) 50.00 aliquot 49ul 50.00 aliquot 48.5ul 50.00 aliquot 47ul
add 0.5ul template add 1ul template add 2.5ul template
add 0.5ul R primer add 0.5ul R primer add 0.5ul R primer


Sample IDs IndX
P1C PCR_R.N2Ind1
P2C PCR_R.N2Ind2
P8P2 PCR_R.N2Ind18
NTC PCR_R.N2Ind18


Program

   98C 30sec 
   (98C 10sec -> 62C 20sec-> 72C 30sec) x 12 cycles
   72C 2min  
   15C hold.

- 2ul of PCR products were analyzed in PAGE gel