AlanFung:LabNotes/Capturing/2012-8-10: Difference between revisions
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==Indexing PCR with Kapa SYBR Fast== | ==Indexing PCR with Kapa SYBR Fast== | ||
*Repeat PCR with same condition as first round of captured library in order to be consistance | *Repeat PCR with same condition as first round of captured library in order to be consistance | ||
*Qiaquick column purify the 3 libraries that were taken out at the early stage of 1st round amplification | |||
*Elute with 30ul EB | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> | {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> | ||
|- style="font-size:8pt" align="center" | |- style="font-size:8pt" align="center" |
Revision as of 07:17, 18 August 2012
Capturing Remaining Gimelbrant lab Sample
- Add 2 drops of mineral oil to prevent evaporation
- Samples stored at tall -20C Alan Rack white box red label "Gimelbrant Lab"
ChIPK36me3 | Concentration ng/ul | Vol | Vol of probes | 10X Ampligase Buffer | H2O | Total |
GM12878 | 0.90 | 40.00 | 0.45 | 2 | 7.6 | 50 |
H0 | 1.00 | 40.00 | 0.50 | 2 | 7.5 | 50 |
H7 | 0.60 | 40.00 | 0.30 | 2 | 7.7 | 50 |
H14 | 0.50 | 40.00 | 0.25 | 2 | 7.8 | 50 |
H16 | 0.60 | 40.00 | 0.30 | 2 | 7.7 | 50 |
ChIPK27me3 | Concentration ng/ul | Vol | Vol of probes | 10X Ampligase Buffer | H2O | Total |
GM12878 | 1.20 | 40.00 | 0.60 | 2 | 7.4 | 50 |
H0 | 2.4 | 40.00 | 1.20 | 2 | 6.8 | 50 |
H7 | 1.2 | 40.00 | 0.60 | 2 | 7.4 | 50 |
H14 | 0.5 | 40.00 | 0.25 | 2 | 7.8 | 50 |
H16 | 1.3 | 40.00 | 0.65 | 2 | 7.4 | 50 |
WCE | Concentration ng/ul | Vol | Vol of probes | 10X Ampligase Buffer | H2O | Total |
GM12878 | 35.39 | 5.65 | 2.49 | 2 | 9.9 | 20 |
P10 | 35.39 | 5.65 | 2.49 | 2 | 9.9 | 20 |
DF2 | 35.39 | 5.65 | 2.49 | 2 | 9.9 | 20 |
H0 | 35.39 | 5.65 | 2.49 | 2 | 9.9 | 20 |
H7 | 35.39 | 5.65 | 2.49 | 2 | 9.9 | 20 |
H14 | 35.39 | 5.65 | 2.49 | 2 | 9.9 | 20 |
H16 | 35.39 | 5.65 | 2.49 | 2 | 9.9 | 20 |
cDNA | Concentration ng/ul | Vol | Vol of probes | 10X Ampligase Buffer | H2O | Total |
GM | 38.12 | 5.25 | 2.49 | 2 | 10.3 | 20 |
H0 | 38.12 | 5.25 | 2.49 | 2 | 10.3 | 20 |
H7 | 38.12 | 5.25 | 2.49 | 2 | 10.3 | 20 |
H14 | 38.12 | 5.25 | 2.49 | 2 | 10.3 | 20 |
H16 | 38.12 | 5.25 | 2.49 | 2 | 10.3 | 20 |
gDNA | Concentration ng/ul | Vol | Vol of probes | 10X Ampligase Buffer | H2O | Total |
H7 | 38.12 | 5.25 | 2.49 | 2 | 10.3 | 20 |
H14 | 38.12 | 5.25 | 2.49 | 2 | 10.3 | 20 |
H16 | 38.12 | 5.25 | 2.49 | 2 | 10.3 | 20 |
- When adding regents to the tube make sure you penetrate the oil layer
Store under βCES36K 95C 2min -> -0.2C/sec to 60C -> 60C 24h -> add 1ul/2.5ul(for 50ul rxn) SLN mix (dNTP 5uM, Stoffel 2U/ul, AmpLigase 1U/ul) -> 60C 18h -> 94C 1min -> 37c 1min -> add 2ul(5ul for 50ul rxn) Exo I&III -> 37C 2h -> 90C 5min -> 4C hold
Prepare SLN Mix
- Dilute 1mM dNTP 1 in 100 to 10uM dNTP
SLN Mix for 20ul Rxn | 1x | 16x |
dNTP (10uM) | 0.5 | 8 |
Stoffel (10U/ul) | 0.2 | 3.2 |
Ampligase (5U/ul) | 0.2 | 3.2 |
H2O | 0.1 | 1.6 |
SLN Mix for 50ul Rxn | 1x | 11x |
dNTP (10uM) | 1.25 | 13.75 |
Stoffel (10U/ul) | 0.5 | 5.5 |
Ampligase (5U/ul) | 0.5 | 5.5 |
H2O | 0.25 | 2.75 |
Prepare Exo I and Exo III Mix
Exo I & III for 20ul Rxn | 1X | 16x |
Exo I (20U/ul) | 1 | 16 |
ExoIII (200U/ul) | 1 | 16 |
Exo I & III for 50ul Rxn | 1X | 11x |
Exo I (20U/ul) | 2.5 | 6.25 |
ExoIII (200U/ul) | 2.5 | 6.25 |
Indexing qPCR
- Perform one step qPCR using Indexing seq. primer
Content (50ul Capture) 4 rxn | Volume | Master Mix | ChIPK36me3 | Index | ChIPK27me3 | Index | ||
Captured DNA | 27 | Do not add to MM | GM12878 | 1 | GM12878 | 10 | ||
2X Kapa HiFi mix | 50 | 550 | H0 | 2 | H0 | 11 | ||
10uM AmpF6.3Sol | 4 | 44 | H7 | 3 | H7 | 12 | ||
10uM AmpR6.3Ind | 4 | Do not add to MM | H14 | 4 | H14 | 13 | ||
H2O | 15 | 165 | H16 | 9 | H16 | 14 | ||
SYBR Green 50X | 0.4 | 4.4 | ||||||
Content (20ul Capture) 7rxn | Volume | Master Mix | WCE | Index | cDNA | Index | gDNA | Index |
Captured DNA | 11 | Do not add to MM | GM12878 | 15 | GM | 22 | H7 | 27 |
2X Kapa SYBG qPCR master mix | 50 | 825 | P10 | 16 | H0 | 23 | H14 | 28 |
10uM AmpF6.3Sol | 4 | 66 | DF2 | 17 | H7 | 24 | H16 | 29 |
10uM AmpR6.3Ind | 4 | Do not add to MM | H0 | 18 | H14 | 25 | ||
H2O | 31 | 511.5 | H7 | 19 | H16 | 26 | ||
SYBR Green 50X | 0.4 | 6.6 | H14 | 20 | ||||
H16 | 21 |
95C 2min -> (98C 20S -> 58C 20S -> 72C 20S) x 6 -> (98C 10S -> 72C 20S) x40 Monitor the reactions and terminate the program right before the amplification curves reach the plateau.
Indexing PCR for 3 samples
Indexing PCR with Kapa SYBR Fast
- Repeat PCR with same condition as first round of captured library in order to be consistance
- Qiaquick column purify the 3 libraries that were taken out at the early stage of 1st round amplification
- Elute with 30ul EB
Content (50ul Capture) 4 rxn | Volume | Master Mix | ChIPK36me3 | Index | ChIPK27me3 | Index | ||
Captured DNA | 27 | Do not add to MM | GM12878 | 1 | GM12878 | 10 | ||
2X Kapa HiFi mix | 50 | 495 | H0 | 2 | H0 | 11 | ||
10uM AmpF6.3Sol | 4 | 39.6 | H7 | 3 | H7 | 12 | ||
10uM AmpR6.3Ind | 4 | Do not add to MM | H14 | 4 | H14 | 13 | ||
H2O | 15 | 148.5 | H16 | 9 | H16 | 14 | ||
SYBR Green 50X | 0.4 | 3.96 | ||||||
Content (20ul Capture) 7rxn | Volume | Master Mix | WCE | Index | cDNA | Index | gDNA | Index |
Captured DNA | 11 | Do not add to MM | GM12878 | 15 | GM | 22 | H7 | 27 |
2X Kapa SYBG qPCR master mix | 50 | 715 | P10 | 16 | H0 | 23 | H14 | 28 |
10uM AmpF6.3Sol | 4 | 57.2 | DF2 | 17 | H7 | 24 | H16 | 29 |
10uM AmpR6.3Ind | 4 | Do not add to MM | H0 | 18 | H14 | 25 | ||
H2O | 31 | 443.3 | H7 | 19 | H16 | 26 | ||
SYBR Green 50X | 0.4 | 5.72 | H14 | 20 | ||||
H16 | 21 | |||||||
Content (15ul Capture) 3rxn | Volume | Master Mix | Type | Sample | Index | |||
Captured DNA | 15 | Do not add to MM | ChIPK36me3 | H0 | 2 | |||
2X Kapa SYBG qPCR master mix | 50 | 165 | WCE | DF2 | 17 | |||
10uM AmpF6.3Sol | 4 | 13.2 | cDNA | GM | 22 | |||
10uM AmpR6.3Ind | 4 | Do not add to MM | ||||||
H2O | 27 | 89.1 | ||||||
SYBR Green 50X | 0.4 | 1.32 |
- The 3 samples highlighted in red will use 15ul from the 1st round PCR (purified product) as the 2nd round PCR is already amplified with Kapa HiFi.
- First add Index primer
- Then add captured DNA
- Prepare Master Mix
98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 6 -> (98C 10S -> 72C 20S) x40 Monitor the reactions and terminate the program right before the amplification curves reach the plateau.