Noi/NOTES/2014-5-13

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BSPP capture of amplified DNA from Illumina[edit]

Sample list:

  • 13 samples received on 2014-03-18 [[1]]. Note: I processed only A1-A10 samples which are amplified DNA samples.
  • 8 samples received on 2014-04-14[[2]]

2014-04-07[edit]

BSPP capture round1 using Polymerase from Illumina[edit]

  • 2014-05-05: Received ~30ul of polymerase with property similar to Amplitaq Stoffel fragment. Conc. 3U/ul. She is not allowed to provide any further info. of this enzyme.
  • I performed BSPP capture on samples A1-A10 and sample #1-#8
Tube ID Conc. (ng/ul) Volume for 200/300ng (ul) Tube ID H2O (ul) GP1V4 probe 10X AmpLigase Buffer Total (ul) Total amount (ng)
A1 189.77 1.58 A1 14.16 2.26 2.00 20.00 300.00
A2 192.53 1.56 A2 14.18 2.26 2.00 20.00 300.00
A3 142.63 2.10 A3 13.64 2.26 2.00 20.00 300.00
A4 145.67 2.06 A4 13.68 2.26 2.00 20.00 300.00
A5 182.27 1.65 A5 14.09 2.26 2.00 20.00 300.00
A6 164.77 1.82 A6 13.92 2.26 2.00 20.00 300.00
A7 112.93 2.66 A7 13.08 2.26 2.00 20.00 300.00
A8 119.33 2.51 A8 13.23 2.26 2.00 20.00 300.00
A9 188.90 1.59 A9 14.15 2.26 2.00 20.00 300.00
A10 205.10 1.46 A10 14.28 2.26 2.00 20.00 300.00
NTC 0.00 0.00 NTC 15.74 2.26 2.00 20.00 0.00
#1 34.6 5.78 #1 9.96 2.26 2.00 20.00 200.00
#2 30.4 6.58 #2 9.16 2.26 2.00 20.00 200.00
#3 39.9 5.01 #3 10.73 2.26 2.00 20.00 200.00
#4 41.4 4.83 #4 10.91 2.26 2.00 20.00 200.00
#5 21.6 9.26 #5 6.48 2.26 2.00 20.00 200.00
#6 30.9 6.47 #6 9.27 2.26 2.00 20.00 200.00
#7 36.1 5.54 #7 10.20 2.26 2.00 20.00 200.00
#8 34.4 5.81 #8 9.93 2.26 2.00 20.00 200.00
NTC 0 0.00 NTC 15.74 2.26 2.00 20.00 0.00
GP1V4 reaction mix[edit]
Components Volume (ul) 22 rxn mix
10X AmpLigase Buffer 2.00 44.00
GP1_V4 (5.75ng/ul) 2.26 49.74
Toal 4.26
Program
-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 16h 
-> add 3ul SLN mix (1.5U/ul Illumina's polymerase (3U/reaction); 0.5U/ul AmpLigase; 100uM dNTP)
-> 55C 6h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.

SLN Mix[edit]

Components Stock conc. Unit Final conc. Unit Prepare volume (ul), total 48ul
Illumina's Polymerase 3 U/ul 1.5 U/ul 24.00
AmpLigase 5 U/ul 0.5 U/ul 4.80
dNTP 1 mM 100 uM 4.80
10x AmpLigase Buffer 10 x 1 x 4.80
H2O 9.60

Amplification[edit]

  • Add 15% v/v captured DNA
Components 1x rxn 22 rxn mix
Captured template 15.00 0.00
10uM AmpF6.4Sol 2.00 44.00
10uM AmpR6.3.IndX (X=1-16) 2.00 0.00
2X KAPA SYBR MM 50.00 1100.00
H2O 31.00 682.00
Total 100.00
- Aliquot 83, add 2ul of AmpR6.3.IndX, 15ul of template
- Use the same PCR profile for library-free protocol
- Stopped reaction at 21 cycles
Samples Index Samples Index Samples Index
A1 Ind_41 A9 Ind_49 #1 Ind_57
A2 Ind_42 A10 Ind_50 #2 Ind_58
A3 Ind_43 NTC-1 Ind_51 #3 Ind_59
A4 Ind_44 NTC-2 Ind_52 #4 Ind_60
A5 Ind_45 NTC-PCR Ind_53 #5 Ind_61
A6 Ind_46 #6 Ind_62
A7 Ind_47 #7 Ind_63
A8 Ind_48 #8 Ind_64
  • Verified PCR product in 6% TBE gel by loading 4ul of PCR product

File:ZhangLab 2 2014-05-09 15hr 25min ILMN GP1V4 gel1 A1-A10 NTC.jpg

File:ZhangLab 2 2014-05-09 15hr 27min ILMN GP1V4 gel2 no1-8 NTC.jpg

  • Perform AMPure bead purification (0.7X volume)
  • Elute with 55ul EB buffer
  • Verify and quantify DNA concentration in 6% TBE gel by loading 4ul of bead-purified DNA

File:ZhangLab 2 2014-05-17 17hr 52min PQ4ul A1-A10 Exp5-07.jpg

File:ZhangLab 2 2014-05-17 17hr 52min PQ4ul no1-no8 NTC Exp5-07.jpg

BSPP capture round2 using Stoffel Fragment[edit]

  • I repeated experiment on samples A1-A10 using Stoffel fragment
  • Everything was the same except I increase amount of template to 400ng since I notice smaple A1-A10 had fainter intensity comparing to sample #1-#8
- Use the same PCR profile for library-free protocol
- Stopped reaction at 22 cycles
  • Verified PCR product in 6% TBE gel by loading 4ul of PCR product

File:ZhangLab 2 2014-05-16 18hr 43min ILMN GP1v4 A1-A8 4ulPCR.jpg

File:ZhangLab 2 2014-05-16 18hr 43min ILMN GP1v4 A9-A10 4ulPCR.jpg

  • Perform AMPure bead purification (0.7X volume)
  • Elute with 55ul EB buffer
  • Verify and quantify DNA concentration in 6% TBE gel by loading 3ul of bead-purified DNA

File:ZhangLab 2 2014-05-17 17hr 52min PQ 3ul A1-A10 Exp5-13.jpg