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