Andrew:Notebook/AR150702 C1
Jump to navigation
Jump to search
Planning
- Sequential MDA on C1 with PEO1
- July 2, 2015
- Experiment ID: AR150702_C1
' | (bases) | ' | ' | ' |
haploid length | 3,100,000,000 | |||
diploid length | 6,200,000,000 | |||
denature length | 12,400,000,000 | |||
phi29 mean processivity | 70,000 | |||
N9 combinations | 262,144 | N6 | ||
% annealed template at 30 C | 98% | 2% | ||
desired gain | 100 | 1000 | ||
space between primers | 691 | 61 | ||
# N9s to cover every base | 1,377,777,778 | |||
# N9s for spacing | 1,968,254 | |||
mols of N9 desired | 3.27E-18 | |||
volume of first MDA (nL) | 31.5 | |||
volume of first MDA (L) | 3.15E-08 | |||
concentration of N9 (M) | 1.038E-10 | |||
concentration of N9 (nM) | 0.1038 | 1 | ||
fold change vs 50 uM | 481,721 | |||
primers (uM) | 50 | |||
primers (M) | 0.00005 | |||
C1 31 nL | Microwell 12 nL | volume (L) | 1.20E-08 | |
dNTPs (mM) | 4.00E-01 | |||
dNTPs (M) | 4.00E-04 | |||
mol dNTPs | 1.26E-11 | |||
# dNTPs | 7.59E+12 | |||
mass/dNTPS incorporated (g/mol) | 3.30E+02 | |||
mass of dntps (g) | 4.16E-09 | |||
mass of dntps (pg) | 4.16E+03 | |||
mass of single genome (pg) | 6.6 | |||
gain | 630 | 240 | ||
Protocol
C1 setup
File:AR150702 reagents-page-001.jpg File:Protocol Worksheet AR150702 C1 v1-page-001.jpg File:Protocol Worksheet AR150702 C1 v1-page-002.jpg File:Protocol Worksheet AR150702 C1 v1-page-003.jpg File:AR150702 thermalProfile.jpg
Off chip
Prep
- Add 1.2 uL 50X SYBR Green I to 120 uL NPM
Nextera XT
- Same as standard protocol, but add 15.5 uL NPM/SYBR Green I mix and run on qPCR machine (final volume 50.5 uL)
0.1X Tn5-059
- 5 uL total
- 3 ul template
- 1 ul SSB
- 1 ul 1X Tn5-059
- Incubate 55 C for 5 min, transfer to cold block
- Add 1 ul 0.2 mg/ml Qiagen Protease
- Incubate 50 C for 15 min, followed by heat inactivation at 70 C for 20 min
- Add 19 uL H2O to bring volume up from 6 uL to 25 uL
- Add 5 uL each index (N7xx/S5xx)
- Add 15.5 uL NPM/SYBR Green I mix
qPCR protocol
- 72 C for 3 min
- 95 C for 1 min
- 25 cycles (but prepare to stop early)
- 95 C for 15 sec
- 55 C for 30 sec
- 72 C for 30 sec, acquisition
- 72 C for 15 sec
- 72 C for 5 min
Results
C1 capture
Chamber | Well | Number | LiveOrDead | Notes |
Ch01 | A03 | 0 | na | na |
Ch02 | A02 | 0 | na | na |
Ch13 | C03 | 1 | both | na |
Ch04 | A09 | 0 | na | na |
Ch05 | A08 | 0 | na | na |
Ch06 | A07 | 0 | na | na |
Ch07 | B03 | 0 | na | na |
Ch08 | B02 | 0 | na | na |
Ch14 | C02 | 1 | both | na |
Ch10 | B09 | 0 | na | na |
Ch11 | B08 | 0 | na | na |
Ch12 | B07 | 0 | na | na |
Ch18 | C07 | 1 | both | na |
Ch24 | D07 | 1 | both | na |
Ch15 | C01 | 0 | na | na |
Ch16 | C09 | 0 | na | na |
Ch25 | E01 | 1 | both | na |
Ch26 | E02 | 1 | both | na |
Ch19 | D03 | 0 | na | na |
Ch20 | D02 | 0 | na | na |
Ch29 | E08 | 1 | both | na |
Ch30 | E09 | 1 | both | na |
Ch23 | Ch | 0 | na | na |
Ch31 | F01 | 1 | both | na |
Ch34 | F07 | 3 | both | na |
Ch35 | F08 | 1 | both | na |
Ch27 | E03 | 0 | na | na |
Ch28 | E07 | 0 | na | na |
Ch36 | F09 | 1 | both | na |
Ch40 | G07 | 1 | both | na |
Ch45 | H03 | 1 | both | extravasating |
Ch32 | F02 | 0 | na | na |
Ch33 | F03 | 0 | na | na |
Ch48 | H09 | 1 | both | extravasating |
Ch17 | C08 | 2 | dead | na |
Ch21 | D01 | 1 | dead | na |
Ch37 | G01 | 1 | dead | na |
Ch38 | G02 | 0 | na | na |
Ch39 | G03 | 0 | na | na |
Ch44 | H02 | 1 | dead | na |
Ch46 | H07 | 1 | dead | na |
Ch42 | G09 | 0 | na | na |
Ch43 | H01 | 0 | na | na |
Ch47 | H08 | 1 | dead | na |
Ch03 | A01 | 1 | live | na |
Ch09 | B01 | 1 | live | na |
Ch22 | D09 | 1 | live | weak |
Ch41 | G08 | 1 | live | na |
Ch49 | A04 | 0 | na | na |
Ch50 | A05 | 0 | na | na |
Ch51 | A06 | 0 | na | na |
Ch52 | A10 | 0 | na | na |
Ch53 | A11 | 0 | na | na |
Ch54 | A12 | 0 | na | na |
Ch55 | B04 | 0 | na | na |
Ch56 | B05 | 0 | na | na |
Ch57 | B06 | 0 | na | na |
Ch58 | B10 | 0 | na | na |
Ch59 | B11 | 0 | na | na |
Ch60 | B12 | 0 | na | na |
Ch61 | C04 | 0 | na | na |
Ch62 | C05 | 0 | na | na |
Ch63 | C06 | 0 | na | na |
Ch64 | C10 | 0 | na | na |
Ch65 | C11 | 0 | na | na |
Ch66 | C12 | 0 | na | na |
Ch67 | D04 | 0 | na | na |
Ch68 | D05 | 0 | na | na |
Ch69 | D06 | 0 | na | na |
Ch70 | D10 | 0 | na | na |
Ch71 | D11 | 0 | na | na |
Ch72 | D12 | 0 | na | na |
Ch73 | E06 | 0 | na | na |
Ch74 | E05 | 0 | na | na |
Ch75 | E04 | 0 | na | na |
Ch76 | E12 | 0 | na | na |
Ch77 | E11 | 0 | na | na |
Ch78 | E10 | 0 | na | na |
Ch79 | F06 | 0 | na | na |
Ch80 | F05 | 0 | na | na |
Ch81 | F04 | 0 | na | na |
Ch82 | F12 | 0 | na | na |
Ch83 | F11 | 0 | na | na |
Ch84 | F10 | 0 | na | na |
Ch85 | G06 | 0 | na | na |
Ch86 | G05 | 0 | na | na |
Ch87 | G04 | 0 | na | na |
Ch88 | G12 | 0 | na | na |
Ch89 | G11 | 0 | na | na |
Ch90 | G10 | 0 | na | na |
Ch91 | H06 | 0 | na | na |
Ch92 | H05 | 0 | na | na |
Ch93 | H04 | 0 | na | na |
Ch94 | H12 | 0 | na | na |
Ch95 | H11 | 0 | na | na |
Ch96 | H10 | 0 | na | debris (clog) |
Gels
File:AR150702 C1 gelsSlide1.png
File:AR150702 C1 gelsSlide2.png
File:AR150702 C1 gelsSlide3.png
File:AR150702 C1 gelsSlide4.png
Sequencing
First round of sequencing
Sample ID | Index Sequence | # of Reads | Run Type, Minmum BP | Library Size | Read 1 Sequencing Primer | Index Primer | Read 2 Sequencing Primer | Notes |
INITIAL-PROJECT-SAMPLE-DATE-Index | CGTGCA | Million | SE/PE, 36+6+36 | SIZE RANGE (BP) | Name | Name | Name | |
AR-150702-D01-NxtXT-N702-S502 | CGTACTAG-CTCTCTAT | 1.5 | SE50+8+8 | 200-800 | HP10 | HP12 | NA | DT Tn5 Old |
AR-150702-E01-NxtXT-N702-S503 | CGTACTAG-TATCCTCT | 1.5 | SE50+8+8 | 200-800 | HP10 | HP12 | NA | DT Tn5 Old |
AR-150702-G01-NxtXT-N702-S505 | CGTACTAG-GTAAGGAG | 1.5 | SE50+8+8 | 200-800 | HP10 | HP12 | NA | DT Tn5 Old |
AR-150702-D01-CoRE-NEBNext25 | TACTGC | 1.5 | SE50+8+8 | 200-800 | HP10 | HP12 | NA | DT Tn5 Old |
AR-150702-E01-CoRE-NEBNext26 | TCGTGA | 1.5 | SE50+8+8 | 200-800 | HP10 | HP12 | NA | DT Tn5 Old |
AR-150702-G01-CoRE-NEBNext27 | TACGCG | 1.5 | SE50+8+8 | 200-800 | HP10 | HP12 | NA | DT Tn5 Old |
AR-150702-D01-Tn5059-N701-S506 | TAAGGCGA-ACTGCATA | 3 | SE50+8+8 | 200-800 | HP10 | HP12 | NA | DT Tn5 New |
AR-150702-E01-Tn5059-N701-S507 | TAAGGCGA-AAGGAGTA | 3 | SE50+8+8 | 200-800 | HP10 | HP12 | NA | DT Tn5 New |
AR-150702-G01-Tn5059-N701-S508 | TAAGGCGA-CTAAGCCT | 3 | SE50+8+8 | 200-800 | HP10 | HP12 | NA | DT Tn5 New |
2nd Round of sequencing
SampleID | Index Sequence | # of Reads (M) | Run Type, Minmum BP | Size range (bp) | Read 1 Primer | Index 1 Primer | Read 2 Primer | Tube labeled |
AR-150702-Ch03-A01-N703-S502 | AGGCAGAA-CTCTCTAT | 2 | SE50+8+8 | 200-800 | HP10 | HP12 | NA | AR150714_Pool1 |
AR-150702-Ch09-B01-N703-S503 | AGGCAGAA-TATCCTCT | 2 | SE50+8+8 | 200-800 | HP10 | HP12 | NA | AR150714_Pool1 |
AR-150702-Ch22-D09-N703-S505 | AGGCAGAA-GTAAGGAG | 2 | SE50+8+8 | 200-800 | HP10 | HP12 | NA | AR150714_Pool1 |
AR-150702-Ch44-H02-N703-S506 | AGGCAGAA-ACTGCATA | 2 | SE50+8+8 | 200-800 | HP10 | HP12 | NA | AR150714_Pool1 |
AR-150702-Ch45-H03-N703-S507 | AGGCAGAA-AAGGAGTA | 2 | SE50+8+8 | 200-800 | HP10 | HP12 | NA | AR150714_Pool2 |
AR-150702-Ch46-H07-N703-S508 | AGGCAGAA-CTAAGCCT | 2 | SE50+8+8 | 200-800 | HP10 | HP12 | NA | AR150714_Pool2 |
AR-150702-Ch47-H08-N703-S504 | AGGCAGAA-AGAGTAGA | 2 | SE50+8+8 | 200-800 | HP10 | HP12 | NA | AR150714_Pool2 |
AR-150702-Ch48-H09-N703-S517 | AGGCAGAA-GCGTAAGA | 2 | SE50+8+8 | 200-800 | HP10 | HP12 | NA | AR150714_Pool2 |
AR-150702-D01-NxtXT-N702-S502 | CGTACTAG-CTCTCTAT | 0.67 | SE50+8+8 | 200-800 | HP10 | HP12 | NA | DT Tn5 Old |
AR-150702-E01-NxtXT-N702-S503 | CGTACTAG-TATCCTCT | 0.67 | SE50+8+8 | 200-800 | HP10 | HP12 | NA | DT Tn5 Old |
AR-150702-G01-NxtXT-N702-S505 | CGTACTAG-GTAAGGAG | 0.67 | SE50+8+8 | 200-800 | HP10 | HP12 | NA | DT Tn5 Old |
AR-150702-D01-CoRE-NEBNext25 | TACTGC | 0.67 | SE50+8+8 | 200-800 | HP10 | HP12 | NA | DT Tn5 Old |
AR-150702-E01-CoRE-NEBNext26 | TCGTGA | 0.67 | SE50+8+8 | 200-800 | HP10 | HP12 | NA | DT Tn5 Old |
AR-150702-G01-CoRE-NEBNext27 | TACGCG | 0.67 | SE50+8+8 | 200-800 | HP10 | HP12 | NA | DT Tn5 Old |