Revision as of 22:35, 2 May 2015 by >Mzcai
in vitro Capture with CA12k_Nov2014 Probe Set + Supp Oligos[edit]
HBRR cDNA Synthesis[edit]
- Followed NEB E6300S protocol: [1]
- Human Brain Reference RNA: [2]
- Labeled concentration = 1ug/ul
- 2 tubes for RNA sample and 1 tube with no RNA (replace with H2O) as negative control
Components
|
Volume
|
RNA |
0.5 ul (0.5 ug)
|
d(T)23VN (50 µM) |
2 ul
|
H2O |
5.5 ul
|
Total |
8 ul
|
- Denatured RNA for 5 min at 70C and then put on ice
- Added the following to tubes
Components
|
Volume
|
M-MuLV Reaction Mix |
10 ul
|
M-MuLV Enzyme Mix |
2 ul
|
Total |
20 ul
|
- Incubated for one hour at 42C
- Inactivated enzyme for 5 min at 80C
- Purified cDNA in one Qiagen PCR clean up column and NTC in another
- Nanodrop w/ dsDNA setting:
- cDNA: 30.7 ng/ul
- Neg Ctrl: 10.2 ng/ul
Calculate Probes Needed[edit]
Probe:target
|
1000:1
|
'
|
Probe size |
3514 |
probes
|
DNA templet |
300 |
ng
|
gDNA MW |
1.95x10^12 |
g/mol
|
gDNA (300ng) |
1.5385x10^-19 |
mol
|
Probe (1000:1) |
1.5385x10^-16 |
mol
|
Probe MW (3514, 150nt) |
1.71585106x10^8 |
g/mol
|
Amount Probe req'd |
26.4 |
ng
|
Probe:target
|
1000:1
|
'
|
Probe size |
2486 |
probes
|
DNA templet |
300 |
ng
|
gDNA MW |
1.95x10^12 |
g/mol
|
gDNA (300ng) |
1.5385x10^-19 |
mol
|
Probe (1000:1) |
1.5385x10^-16 |
mol
|
Probe MW (2486, 150nt) |
1.21388894x10^8 |
g/mol
|
Amount Probe req'd |
18.7 |
ng
|
Probes, Target and Ampligase Buffer Mix[edit]
- gDNA: 12878 (80.3ng/ul)
- First strand cDNA from HBRR
- CA12k_Nov2014 Probes
- V4 819nM x (150nt*325Da/nt + 79Da) x 10^-6 L/ul = 40.0 ng/ul
- V7 768nM x (150nt*325Da/nt + 79Da) x 10^-6 L/ul = 37.5 ng/ul
- Diluted 1:2 with H2O for easier pipetting (final conc 20.0ng/ul and 18.75ng/ul)
- NTC is the NTC from first strand cDNA synthesis
- 5nM of each supp oligos (5 x 10^-6 nmol/ul)
- 100-fold more of each suppressor oligo = 1.5385 x 10^-14 mol = 15 x 10^-6 nmol
Sample #
|
Sample Description
|
Probes
|
Target
|
5nM Supp Oligos
|
10X Ampligase Buffer
|
H2O
|
Total
|
1 |
V4 - gDNA |
1.3 |
3.8 |
3 |
3 |
18.9 |
30
|
2 |
V4 - cDNA |
1.3 |
15 |
3 |
3 |
7.7 |
30
|
3 |
V4 - NTC |
1.3 |
15 |
3 |
3 |
7.7 |
30
|
4 |
V7 - gDNA |
1 |
3.8 |
3 |
3 |
19.2 |
30
|
5 |
V7 - cDNA |
1 |
15 |
3 |
3 |
8 |
30
|
6 |
V7 - NTC |
1 |
15 |
3 |
3 |
8 |
30
|
- Sample 4, accidentally added an extra 9ul H2O so added 4ul 10X Ampligase Buffer
- Added 4ul of AmpLigase enzyme mix as well (vs 3ul for others)
- Add 40ul mineral oil on top
Program
- 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 20h
- -> add 3ul AmpLigase enzyme mix (0.5U/ul AmpLigase in 1X AmpLigase buffer)
- -> 55 C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
AmpLigase enzyme mix[edit]
Components
|
Stock conc.
|
Unit
|
Final conc.
|
Unit
|
Prepare volume 30ul
|
AmpLigase |
5 |
U/ul |
0.5 |
U/ul |
2.00
|
10x AmpLigase Buffer |
10 |
x |
1 |
x |
2.00
|
H2O |
|
|
|
|
16.00
|
Total |
|
|
|
|
20.00
|
Add Sequence Adapters PCR[edit]
Primers[edit]
Primer
|
Sequence
|
Index #
|
ISB_CA_AF |
AATGATACGGCGACCACCGAGATCTACACGCCTGCATATCGGGAAGCTGAAG |
|
ISB_CA_AR.T1 |
CAAGCAGAAGACGGCATACGAGATCGTGATCGGTCTGCCTTCCCGATATCCGACGG |
Indx1
|
ISB_CA_AR.T2 |
CAAGCAGAAGACGGCATACGAGATACATCGCGGTCTGCCTTCCCGATATCCGACGG |
Indx2
|
ISB_CA_AR.T3 |
CAAGCAGAAGACGGCATACGAGATGCCTAACGGTCTGCCTTCCCGATATCCGACGG |
Indx3
|
Sample
|
Index
|
Forward Primer
|
Reverse Primer
|
1 |
1 |
ISB_CA_AF |
ISB_CA_AR.T1
|
2 |
2 |
ISB_CA_AF |
ISB_CA_AR.T2
|
3 |
3 |
ISB_CA_AF |
ISB_CA_AR.T3
|
4 |
1 |
ISB_CA_AF |
ISB_CA_AR.T1
|
5 |
2 |
ISB_CA_AF |
ISB_CA_AR.T2
|
6 |
3 |
ISB_CA_AF |
ISB_CA_AR.T3
|
PCR Test[edit]
Components
|
1X Volume
|
6.5X Volume
|
Captured template |
1 |
0
|
10uM Forward Primer |
0.4 |
2.6
|
10uM Reverse Primer |
0.4 |
0
|
2X KAPA SYBG MM |
12.5 |
81.25
|
H2O |
10.7 |
69.55
|
Total |
25 |
153.4
|
- Aliquot 23.6ul from 6.5X master mix and add 1ul captured template and 0.4ul corresponding reverse primer
Program
98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min
File:20150501 CA12kNov2014 invitroPCRtest.JPG
- Looks good; NTC are both negative so I won't amplify on next PCR
Components
|
1X Volume
|
4.5X Volume
|
Captured template |
12 |
0
|
10uM Forward Primer |
2 |
9
|
10uM Reverse Primer |
2 |
0
|
2X KAPA SYBG MM |
50 |
225
|
H2O |
34 |
153
|
Total |
100 |
387
|
- Aliquot 86ul from 4.5X master mix and add 12ul captured template and 2ul corresponding reverse primer
Program
98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x13 -> 72C 3min
File:20150502 CA12kNov2014 invitroPCR.JPG
- Bead purification with 1.5:1 Beads to amplicon volume ratio
- Eluted with 50ul total for each sample
PAGE Check[edit]
- Load 2ul of each sample + 2ul loading dye
File:2015-05-02 CA12kNov2014 invitro gelcheck.jpg
- Labeled tubes and put in "Non-quantified Sequencing Libraries"
- Sample1: MC20150502_CA12kNov14supp_V4gDNA-1
- Sample2: MC20150502_CA12kNov14supp_V4cDNA-2
- Sample3: MC20150502_CA12kNov14supp_V7gDNA-4
- Sample4: MC20150502_CA12kNov14supp_V7cDNA-5