Noi/NOTES/2014-5-23

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BSPP capture of amplified DNA from Illumina using 3 different enzymes for polymerization[edit]

[Link to calendar]

  • From previous experiment on 2014-05-13 [[1]], the results were inconclusive, and I lacked positive control. Dr. Zhang suggested to repeat experiment on few samples from Illumina and include cancer sample DNA as control.
  • I need to prepare more GP1V4 BSPP.

Sample list and experimental design[edit]

  • Cancer sample #1 or C1 (BXPC3)
  • Cancer sample #2 or C2 (U87MG)
  • Illumina amplified DNA, A1
  • Illumina amplifed DNA, A2
  • Capture with 500:1 GP1V4:target ratio (total amount in each reaction ~13ng)
  • Bisulfite treated DNA/amplified DNA amount 200ng
  • Add different enzyme in SLN mix,
    • Stoffel (blue color)
    • Hemo KlenTaq (red color)
    • Illumina Polymerase (black color)
  • I include sample C1, A1, and A2 in capture reaction, but not include GP1V4. This is may not necessary, but I want to make sure that there is no any background from the template itself.
  • For cancer samples, I performed bisulfite conversion 6tubes of C1 and 3tubes of C2 samples (600ng gDNA for each tube). I used bisulfite treated DNA from different tubes for each reaction. There might me some variations of DNA amount in each reaction based on accuracy of DNA concentration quantification or pipetting.

Bisulfite conversion[edit]

  • I did bisulfite converssion by usingEZ DNA Methylation-Lightning™ Kit -Zymo and followed the protocol of the kit
  • Follow the instruction of the kit
 Program 

1. 98°C for 8 minutes
2. 54°C for 60 minutes
3. 4°C hold
  • Elute with 16ul elution buffer. Total volume left after elution ~15ul
  • Took 2ul for Qubit ssDNA assay
Sample Concentration in the Qubit ' uL used Dilution Sample Concentration ' Yield in 15ul (ng) % Recovery
CANC1-1 219 ng/mL 2 100 21.9 ug/mL 328.50 54.75
CANC1-2 204 ng/mL 2 100 20.4 ug/mL 306.00 51.00
CANC1-3 231 ng/mL 2 100 23.1 ug/mL 346.50 57.75
CANC1-4 209 ng/mL 2 100 20.9 ug/mL 313.50 52.25
CANC1-5 226 ng/mL 2 100 22.6 ug/mL 339.00 56.50
CANC1-6 227 ng/mL 2 100 22.7 ug/mL 340.50 56.75
CANC2-1 207 ng/mL 2 100 20.7 ug/mL 310.50 51.75
CANC2-2 195 ng/mL 2 200 19.5 ug/mL 292.50 48.75
CANC2-3 209 ng/mL 2 100 20.9 ug/mL 313.50 52.25

BSPP capture set up[edit]

  • BSC_A1 conc. = 120ng/ul
  • BSC_A2 conc. = 100ng/ul
  • GP1V4 conc. (prep 2014-05-20) = 8.37ng/ul

Probe & AmpLigase Buffer mix[edit]

Mix I. Amligase buffer and GP1V4[edit]
Components 1 rxn 18 rxn mix
GP1V4 (8.37ng/ul) 1.55 27.96
10X AmpLig Buffer 2.00 36.00
H2O 5.00 90.00
Total 8.55
Mix II. only Amligase buffer[edit]
Components 1 rxn 11 rxn mix
10X AmpLig Buffer 2.00 22.00
H2O 5.00 55.00
Total 7.00
Stoffel C1-S C2-S A1-S A2-S NTC C1 no GP1V4 A1 no GP1V4 A2 no GP1V4
Conc. (ng/ul) 21.90 20.70 120.00 100.00 20.40 120.00 100.00
Vol. for 200ng 9.13 9.66 1.67 2.00 0.00 9.80 1.67 2.00
GP1V4 (8.37ng/ul) 1.55 1.55 1.55 1.55 1.55 0.00 0.00 0.00
H2O 2.31 1.78 9.78 9.45 11.45 3.20 11.33 11.00
10X Ampligase buffer 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00
H2O in Mix 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00
Total 20.00 20.00 20.00 20.00 20.00 20.00 20.00 20.00
Hemo Klentaq C1-S C2-S A1-S A2-S NTC C1 no GP1V4 A1 no GP1V4 A2 no GP1V4
Conc. (ng/ul) 23.10 19.50 120.00 100.00 20.90 120.00 100.00
Vol. for 200ng 8.66 10.26 1.67 2.00 0.00 9.57 1.67 2.00
GP1V4 (8.37ng/ul) 1.55 1.55 1.55 1.55 1.55 0.00 0.00 0.00
H2O 2.79 1.19 9.78 9.45 11.45 3.43 11.33 11.00
10X Ampligase buffer 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00
H2O in Mix 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00
Total 20.00 20.00 20.00 20.00 20.00 20.00 20.00 20.00
ILMN-Pol C1-S C2-S A1-S A2-S NTC C1 no GP1V4 A1 no GP1V4 A2 no GP1V4
Conc. (ng/ul) 22.60 20.90 120.00 100.00 22.70 120.00 100.00
Vol. for 200ng 8.85 9.57 1.67 2.00 0.00 8.81 1.67 2.00
GP1V4 (8.37ng/ul) 1.55 1.55 1.55 1.55 1.55 0.00 0.00 0.00
H2O 2.60 1.88 9.78 9.45 11.45 4.19 11.33 11.00
10X Ampligase buffer 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00
H2O in Mix 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00
Total 20.00 20.00 20.00 20.00 20.00 20.00 20.00 20.00
Program
-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h 
-> add 2ul SLN mix (2U/ul Polymerase (NEB); 0.5U/ul AmpLigase; 100uM dNTP)
-> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
  • For Illumina's polymerase I used 1ul for each sample as recommended by Xuyu

SLN mix solution[edit]

Components Stock conc. Unit Final conc. Unit Prepare volume (ul), total 20ul
Stoffel/ Hemo KlenTaq 10 U/ul 2 U/ul 4.00
AmpLigase 5 U/ul 0.5 U/ul 2.00
dNTP 1 mM 100 uM 2.00
10x AmpLigase Buffer 10 x 1 x 2.00
H2O 10.00
Components Stock conc. Unit Final conc. Unit Prepare volume (ul), total 20ul
Illumina's Polymerase 3 U/ul 1.5 U/ul 10.00
AmpLigase 5 U/ul 0.5 U/ul 2.00
dNTP 1 mM 100 uM 2.00
10x AmpLigase Buffer 10 x 1 x 2.00
H2O 4.00

PCR Amplification[edit]

  • I performed PCR on half of captured DNA (10ul) in total 50ul PCR reaction (20% v/v in PCR)
Components 1x rxn 26 rxn mix
Captured template 10.00 0.00
10uM AmpF6.4Sol 1.00 26.00
10uM AmpR6.3.IndX (X=1-24) 1.00 0.00
2X KAPA SYBR MM 25.00 650.00
H2O 13.00 338.00
Total 50.00
  • Aliquot 39ul + 1ul AmpR6.3 IndX + 10ul DNA template
Samples C1 C2 A1 A2 NTC C1 no GP1V4 A1 no GP1V4 A2 no GP1V4
Strip 1: Stoffel Ind_1 Ind_2 Ind_3 Ind_4 Ind_5 Ind_6 Ind_7 Ind_8
Strip 2: Hemo KlenTaq Ind_9 Ind_10 Ind_11 Ind_12 Ind_13 Ind_14 Ind_15 Ind_16
Strip 3: ILMN's Pol Ind_17 Ind_18 Ind_19 Ind_20 Ind_21 Ind_22 Ind_23 Ind_24
Program (Eppendorf Realplex)
 
98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min
  • I stopped all samples at 21x cycles except sample C1,C2 with Hemo KlenTaq/ Illumina' Polymerase at 22x cycles.
  • Verify 3ul of PCR product in 6% TBE gel (no AMPure bead purified)

Stoffel Fragment
File:ZhangLab 2 2014-05-26 13hr 01min GP1v4 C1C2A1A2NTC Stoffel.jpg
Hemo KlenTaq
File:ZhangLab 2 2014-05-26 13hr 01min GP1v4 C1C2A1A2ntc HemoKlenTaq.jpg
Illumina's Polymerase
File:ZhangLab 2 2014-05-26 13hr 01min GP1v4 C1C2A1A2ntc ILPol.jpg

  • ** = over-amplified
  • From the result above, it's clear that cancer samples with Stoffel fragment and Hemo Klentaq showed a smear between 350~390bp. I think there is the variation of DNA input in different tubes because there is more background in the samples with strong signal.
  • Amplified DNA from Illumina showed the same pattern in three enzyme, but has highest background in Hemo KlenTaq
  • With Illumina's polymerase, cancer samples showed discrete bands of ~350, 390 and above 400bp similar to amplified DNA from Illumina.

  • Example of GP1V4 capture on cancer and blood samples I did experiment on 2013-12-24 (after AMPure bead purified)

File:ZhangLab 2 2013-12-25 19hr 44min GP1 V4 1-8 PQ.jpg