Revision as of 23:51, 10 July 2013 by >Mzcai
Verify 20 gap Probe Capture[edit]
- Repeat probe capture from 2013-6-28 due to low capture efficiency of 20 gap probes and peculiar result for NTC reaction
- Capture NTC, gDNA, and cDNA with Agi26k_20gap probes and gDNA with CES36k18bp as positive control
Synthesis of cDNA from 1ug Human Brain Ref RNA[edit]
- Followed NEB E6300S protocol: [1]
- Used 1 ul of Human Brain Reference RNA at concentration 1ug/ul for cDNA synthesis
- Mixed components in 0.2 ml microfuge tube labelled HBRR 1ug cDNA
Components
|
Volume
|
RNA |
1 ul (1 ug)
|
d(T)23VN (50 µM) |
2 ul
|
H2O |
5 ul
|
Total |
8 ul
|
- Denatured RNA for 5 min at 70C and then put on ice
- Added the following to tube
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
- Followed NEB E6111S protocol: [2]
- Added 48 ul H20
- Added 8ul 10x Second strand synthesis reaction buffer
- Added 4ul Second strand synthesis enzyme mix
- Mixed by pipetting
- Incubated for 2.5 hours at 16C
- Purified using Qiagen minelute column (eluted 21ul)
- Measured DNA conc with Nandrop: 47ng/ul
- 19.5 ul (916.5 ng) sample left in tube
Probe Capture[edit]
'
|
Agi26k_20gap (16.79ng/ul)
|
CES36k18bp (75.5ng/ul)
|
'
|
Probe Size |
12964 |
36456 |
|
gDNA 12878 (100ng/ul) |
300 ng |
300 ng |
|
gDNA MW |
1.95x10^12 g/mol |
1.95x10^12 g/mol |
|
gDNA |
1.5385x10^-19 mol |
1.5385x10^-19 mol |
|
Probe Required (1000:1) |
1.5385x10^-16 mol |
1.5385x10^-16 mol |
|
Probe MW |
8.1419x10^8 g/mol |
1.1877x10^9 g/mol |
(Probe size x (Probe length x 325 Da/nt + 79Da)
|
Amount Probe Required |
125 ng (7.44ul) |
183 ng (2.42 ul) |
|
|
20 gap probes from 2013-6-6
Tube #
|
Probe Type
|
Sample
|
Probe (ul)
|
10X Ampligase Buffer
|
Target (ul)
|
H2O
|
Total
|
1 |
Agi26k_20gap |
NTC |
7.44 |
3 |
0 |
19.56 |
30
|
2 |
Agi26k_20gap |
gDNA 12878 |
7.44 |
3 |
3 |
16.56 |
30
|
3 |
Agi26k_20gap |
cDNA |
7.44 |
3 |
19 |
0.56 |
30
|
4 |
CES36k18bp |
gDNA 12878 |
2.42 |
3 |
3 |
21.58 |
30
|
Program
- 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 20h
- -> add 2ul SLN mix (2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 100uM dNTP mix)
- -> 55 C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
SLN mix[edit]
- Noi made the mix (NEED TO UPDATE THIS SECTION WHEN DETAILS ARE UP ON NOI'S WIKI)
- Noi heat activated the Taq polymerase before adding to the mix
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
|
dNTP Mix |
1 |
mM |
100 |
uM |
1.00
|
Titanium Taq DNA |
10 |
U/ul |
2 |
U/ul |
2.00
|
H2O |
|
|
|
|
5.00
|
Total |
|
|
|
|
10.00
|