Revision as of 03:21, 12 July 2017 by >Mzcai
Agi15k_Feb2017 V4 + 5% formamide in vitro Capture[edit]
Calculate Probes Needed[edit]
Probe:target
|
1000:1
|
'
|
Probe size |
4998 |
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 (4998, 157nt) |
2.387x10^8 |
g/mol
|
Amount Probe req'd |
36.7 |
ng
|
Probes, Target and Ampligase Buffer Mix[edit]
Sample
|
Condition
|
V4 (10.3ng/ul)
|
12878 DNA (80.3ng/ul)
|
UHRR (1ug/ul)
|
Ampligase Buffer
|
SplintR Buffer
|
Formamide or DMF
|
Ribolock 40U/ul
|
H2O
|
Total
|
1
|
Ampligase DNA
|
3.6
|
3.8
|
0
|
3
|
0
|
0
|
0
|
19.6
|
30
|
2
|
Ampligase NTC
|
3.6
|
0
|
0
|
3
|
0
|
0
|
0
|
23.4
|
30
|
3
|
SplintR 10% DMF DNA
|
3.6
|
3.8
|
0
|
0
|
3
|
3
|
0
|
16.6
|
30
|
4
|
SplintR 10% DMF RNA
|
3.6
|
0
|
1
|
0
|
3
|
3
|
0.5
|
18.9
|
30
|
5
|
SplintR 10% DMF NTC
|
3.6
|
0
|
0
|
0
|
3
|
3
|
0
|
20.4
|
30
|
6
|
SplintR 5% formamide DNA
|
3.6
|
3.8
|
0
|
0
|
3
|
1.5
|
0
|
18.1
|
30
|
7
|
SplintR 5% formamide RNA
|
3.6
|
0
|
1
|
0
|
3
|
1.5
|
0.5
|
20.4
|
30
|
8
|
SplintR 5% formamide NTC
|
3.6
|
0
|
0
|
0
|
3
|
1.5
|
0
|
21.9
|
30
|
Program
- 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 16h
- Should have added RiboLock after reached 55C since it would be denatured at 95C
- Ampligase: add 3ul AmpLigase enzyme mix (0.5U/ul AmpLigase in 1X AmpLigase buffer)
- SplintR: add 3ul SplintR enzyme mix (10ul SplintR + 2ul 10X SplintR Buffer + 4ul Ribolock + 4ul H2O)
- Ampligase: -> 55 C 20h -> 94C 2min -> add 2ul Exo I/III mix -> 37C 1h -> 94C 2min -> 4C hold
- SplintR: -> 37 C 30min -> 94C 10min -> add 2ul Exo I/III mix -> 37C 1h -> 94C 2 min -> 4C hold
- Take 30ul and purify with Zymo columns
- Left in 4C 1 hour after first column spin down
- Elute 10ul
AmpLigase enzyme mix[edit]
Components
|
Stock conc.
|
Unit
|
Final conc.
|
Unit
|
Prepare volume 10ul
|
AmpLigase |
5 |
U/ul |
0.5 |
U/ul |
1.00
|
10x AmpLigase Buffer |
10 |
x |
1 |
x |
1.00
|
H2O |
|
|
|
|
8.00
|
Total |
|
|
|
|
10.00
|
Quantify[edit]
- qPCR all 8 samples 1ul each with triplicates
Components
|
1X Volume
|
25X Volume
|
Captured template |
1 |
0
|
10uM ISB_CA_AF |
0.4 |
10
|
10uM ISB_CA_AR.T2 |
0.4 |
10
|
2X KAPA SYBG MM |
12.5 |
312.5
|
H2O |
10.7 |
267.5
|
Total |
25 |
600
|
- Aliquot 24ul from 24X master mix and add 1ul captured template
Program
98C 1min -> (98C 10s -> 52C 20s -> 72C 20s)x30 -> 72C 3min
File:20170622 Agi15kFeb17 invitrocapture SplintR5formamide.PNGFile:20170622 Agi15kFeb17 invitrocapture SplintR5formamide Ct.PNG
- No separation between DNA, RNA, and NTC samples for SplintR
- Sequence results to see if there's no difference really
- Stop Ampligase NTC at 28 cycles
- Stop others at 19 cycles
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 |
1 |
ISB_CA_AF |
ISB_CA_AR.T1
|
4 |
2 |
ISB_CA_AF |
ISB_CA_AR.T2
|
5 |
3 |
ISB_CA_AF |
ISB_CA_AR.T3
|
6 |
1 |
ISB_CA_AF |
ISB_CA_AR.T1
|
7 |
2 |
ISB_CA_AF |
ISB_CA_AR.T2
|
8 |
3 |
ISB_CA_AF |
ISB_CA_AR.T3
|
Components
|
1X Volume
|
8.5X Volume
|
Captured template |
5 |
0
|
10uM Forward Primer |
2 |
17
|
10uM Reverse Primer |
2 |
0
|
2X KAPA SYBG MM |
50 |
425
|
H2O |
41 |
348.5
|
Total |
100 |
790.5
|
- Aliquot 93ul from 8.5X master mix and add 5ul captured template and 2ul corresponding reverse primer
Program
98C 1min -> (98C 10s -> 52C 20s -> 72C 20s)x30 -> 72C 3min
- Sample 1: Stopped at 20 cycles
- Sample 2: Stopped at 28 cycles
- Sample 3-5: Stopped at 18 cycles
- Sample 6-8: Stopped at 17 cycles
Excel data with PCR curve
File:2017-07-10 Agi15kFeb17V4 invitroCapture SeqLibrarySizeCheck.jpg
- Only Ampligase NTC band looks different, otherwise every sample has bands of expected size for captured padlock probes with sequencing adapters
- Nanodrop: every sample was ~500ng/ul +/- 10ng/ul
- Mix Samples 1&2, 3&4&5, 6&7&8 in equal ratios (20ul each)
- Gel size selection 3 lanes
- Load each well with 10ul mix (~5ug) + 10ul 6x loading buffer
File:2017-06-27 Agi15kFeb17V4 invitroCapture SeqLibrarySizeSelect.jpg
- Cut out band and shear through 0.5ul tube into 2.0ul tube
- Add 450ul 1X TE Buffer and vortexed at 37C for 1hr
- Centrifuged at 15,000 rpm for 3 min at RT
- Transferred the clear spnt. to Nanosep columns and centrifuged at 15,000 rpm for 3 min
- Transferred the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min
- Transferred spnt to fresh 1.5 mL tube
- Precipitated in 3 1.5ml tubes with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 44ul of 3M NaoAc pH 5.2
- Vortexed and placed the 8 tubes at -80C 30min
- Spun 8 tubes at 10,000rpm at 4C for 30min
- Discard supernatant and add 650ul cold 80% EtOH and spin at 14,000rpm at 4C for 5min
- Discard supernatant and let dry in hood for 10min
- Resuspend each tube with 10ul and combine
For Sequencing[edit]
- MC20170619_Agi15kFeb2017_V4_AmpLigase_Indx1,2
- MC20170619_Agi15kFeb2017_V4_SplintRDMF_Indx1,2,3
- MC20170619_Agi15kFeb2017_V4_SplintRForm_Indx1,2,3