Matt:LabNotes/2016-6-24: Difference between revisions
Jump to navigation
Jump to search
>Mzcai m (→PCR Test) |
>Mzcai mNo edit summary |
||
Line 119: | Line 119: | ||
98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x21 | 98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x21 | ||
[[450px]] | [[File:2016-06-27_TB12kApr2016_V7_intubecapture_testPCR.jpg|450px]] | ||
====PCR==== | ====PCR==== | ||
Line 142: | Line 142: | ||
98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x13 -> 72C 3min -> 4C hold | 98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x13 -> 72C 3min -> 4C hold | ||
[[File:|450px]] | [[File:2016-06-27_TB12kApr2016_V7_intubecapture_PCR.jpg|450px]] | ||
*Bead purification with | *Bead purification with 0.8:1 Beads to amplicon volume ratio | ||
**Eluted with 50ul total for each sample | **Eluted with 50ul total for each sample | ||
===PAGE Check=== | ===PAGE Check=== | ||
[[File: | [[File:|250px]] | ||
*Labeled tubes and put in "Non-quantified Sequencing Libraries" | *Labeled tubes and put in "Non-quantified Sequencing Libraries" | ||
** | **Sample: MC20160628_TB12kApr16V7_gDNA-1 (MC160628_TB12kAprV7_gDNA-1) | ||
Revision as of 22:39, 28 June 2016
in vitro Capture with TB12k_Apr2016 V7
- Will capture gDNA from mouse embryonic fibroblasts (got from Andrew)
Sample Groups
- gDNA MEF (77ng/ul)
- Negative Control
gDNA
Probe:target | 1000:1 | ' |
Probe size | 1808 | probes |
DNA template | 300 | ng |
gDNA MW | 1.8x10^12 | g/mol |
gDNA (300ng) | 1.66x10^-19 | mol |
Probe (1000:1) | 1.66x10^-16 | mol |
Probe MW (1808, 160nt) | 8.7997168x10^7 | g/mol |
Amount Probe req'd | 14.6 | ng |
- 374nM = 18.2ng/ul
Sample # | Sample Description | Probes | Target | 10X Ampligase Buffer | H2O | Total |
1 | gDNA | 0.8 | 3.9 | 3 | 22.3 | 30 |
2 | NTC | 0.8 | 0 | 3 | 26.2 | 30 |
- Add 40ul Mineral Oil on top
Program
- 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 20h
- In BioRad Thermalcycler program says: -0.2C per cycle every 30sec
- -> add 3ul AmpLigase enzyme mix (0.5U/ul AmpLigase in 1X AmpLigase buffer)
- -> 55 C 20h (actually 24h)-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
- Tiffany accidentally stopped reaction after 30min of Exo I/III digestion
- She let it run 2min at 94C and then put it in fridge
- Soon after, she called and I asked her to add 2ul of fresh Exo I/III and finish out the protocol with 37C 2h -> 94C 2min -> 4C hold
AmpLigase enzyme mix
Components | Stock conc. | Unit | Final conc. | Unit | Prepare volume 30ul |
AmpLigase | 5 | U/ul | 0.5 | U/ul | 1 |
10x AmpLigase Buffer | 10 | x | 1 | x | 1 |
H2O | 8 | ||||
Total | 10 |
Add Sequence Adapters PCR
Primers
Primer | Sequence | Index # |
ISB_CA_AF | AATGATACGGCGACCACCGAGATCTACACGCCTGCATATCGGGAAGCTGAAG | |
ISB_CA_AR.T1 | CAAGCAGAAGACGGCATACGAGATCGTGATCGGTCTGCCTTCCCGATATCCGACGG | Indx1 |
ISB_CA_AR.T2 | CAAGCAGAAGACGGCATACGAGATACATCGCGGTCTGCCTTCCCGATATCCGACGG | Indx2 |
Sample | Index | Forward Primer | Reverse Primer |
gDNA | 1 | ISB_CA_AF | ISB_CA_AR.T1 |
NTC | 2 | ISB_CA_AF | ISB_CA_AR.T2 |
PCR Test
Components | 1X Volume | 2.5X Volume |
Captured template | 1 | 0 |
10uM Forward Primer | 0.4 | 1 |
10uM Reverse Primer | 0.4 | 0 |
2X KAPA SYBG MM | 12.5 | 31.25 |
H2O | 10.7 | 26.75 |
Total | 25 | 62.5 |
Program 98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x21
File:2016-06-27 TB12kApr2016 V7 intubecapture testPCR.jpg
PCR
Components | 1X Volume | 2.5X Volume |
Captured template | 12 | 0 |
10uM Forward Primer | 2 | 5 |
10uM Reverse Primer | 2 | 0 |
2X KAPA SYBG MM | 50 | 125 |
H2O | 34 | 85 |
Total | 100 | 250 |
Program 98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x13 -> 72C 3min -> 4C hold
File:2016-06-27 TB12kApr2016 V7 intubecapture PCR.jpg
- Bead purification with 0.8:1 Beads to amplicon volume ratio
- Eluted with 50ul total for each sample
PAGE Check
[[File:|250px]]
- Labeled tubes and put in "Non-quantified Sequencing Libraries"
- Sample: MC20160628_TB12kApr16V7_gDNA-1 (MC160628_TB12kAprV7_gDNA-1)