Revision as of 21:04, 15 March 2013 by >Noi
Capture set up (Matt's project)[edit]
NTC (no targets)
1-1. gDNA (12878)
2-1. Total RNA
3-1. ds cDNA no RNasA treatment
4-1. ds cDNA RNaseA treatment
1-2. gDNA (12878)
2-2. Total RNA
3-2. ds cDNA no RNasA treatment
4-2. ds cDNA RNaseA treatment
Probe calculation[edit]
Probe:target
|
1000:1
|
'
|
Probe size |
12,335 |
probes
|
Template |
300 |
ng
|
Human gDNA MW |
1.9500E+12 |
g/mol (3E+09 * 650Da/bp+157.9Da)
|
Human gDNA (300ng) |
1.5385E-19 |
mol (300ng/1.9500E+12 g/mol)
|
Probe required (1000:1) |
1.5385E-16 |
mol (1.5385E-19mol x 1000)
|
Probe MW (12,335 probes, 170nt) |
6.8151E+08 |
g/mol (12335 probes * 170nt * 325Da/nt + 79Da)
|
Amount probe required |
1.05E-07 |
g (6.8151E+08 g/mol * 1.5385E-16mol)
|
Amount probe required |
105.00 |
ng
|
- Since I resuspend the probes in too large volume, I concentrate using Qiaquick column, elute in total volume ~62ul, re-quantify conc. ~22ng/ul
- Note:
- I showed Matt how to calculate probes and he did calculation, and the number he got agreed with this calculation
- Even the target amounts in each reaction are not the same, I used the same probe amount in every reaction based on gDNA calculation
Probes and AmpLigase Buffer Mix[edit]
Components
|
Volume (ul)
|
9.5 rxn mix
|
12K Insitu probes (22ng/ul) |
4.77 |
45.32
|
10X AmpLigase buffer |
3.00 |
28.50
|
Total |
7.77 |
73.82
|
Samples
|
12K Insitu probes
|
10X AmpLigase buffer
|
Conc. (ng/ul)
|
Targets (ul)
|
H2O (ul)
|
Total (ul)
|
Total amount in capture (ng)
|
NTC |
4.77 |
3.00 |
0 |
0 |
22.23 |
30.00 |
0.00
|
1-1. gDNA (12878) |
4.77 |
3.00 |
45.10 |
6.65 |
15.58 |
30.00 |
299.92
|
2-1. Total RNA |
4.77 |
3.00 |
1000.00 |
1.00 |
21.23 |
30.00 |
1000.00
|
3-1. ds cDNA no RNasA treatment |
4.77 |
3.00 |
15.20 |
19.00 |
3.23 |
30.00 |
288.80
|
4-1. ds cDNA RNaseA treatment |
4.77 |
3.00 |
5.84 |
19.00 |
3.23 |
30.00 |
110.96
|
1-2. gDNA (12878) |
4.77 |
3.00 |
45.10 |
6.65 |
15.58 |
30.00 |
299.92
|
2-2. Total RNA |
4.77 |
3.00 |
1000.00 |
1.00 |
21.23 |
30.00 |
1000.00
|
3-2. ds cDNA no RNasA treatment |
4.77 |
3.00 |
10.20 |
19.00 |
3.23 |
30.00 |
193.80
|
4-2. ds cDNA RNaseA treatment |
4.77 |
3.00 |
7.77 |
19.00 |
3.23 |
30.00 |
147.63
|
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 |
3.00
|
10x AmpLigase Buffer |
10 |
x |
1 |
x |
3.00
|
H2O |
|
|
|
|
24.00
|
Total |
|
|
|
|
30.00
|