Revision as of 20:34, 9 July 2013 by >Mzcai
Verify 20 gap Probe Capture
- 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
- 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 23ul into 1.5ml tube labelled HBRR cDNA 3.11.13)
- Measured DNA conc with Nandrop: ___
Probe Capture
'
|
Agi26k_20gap
|
CES36k18bp
|
'
|
Probe Size |
12964 |
13179 |
|
gDNA HAPMAP C1 (87ng/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 |
8.2769x10^8 g/mol |
(Probe size x (193nt x 325 Da/nt + 79Da)
|
Amount Probe Required |
125 ng (7.44ul) |
127 ng (4.47 ul) |
|
|
0 gap probes from 2013-6-27
20 gap probes from 2013-6-6
cDNA from 2013-6-26
Tube #
|
Probe type
|
Samples
|
Probes (ul)
|
10X AmpLigase Buffer (ul)
|
Target (ul)
|
H2O (ul)
|
Total
|
1 |
0 gap (28.4ng/ul) |
NTC |
4.47 |
3 |
0 |
22.53 |
30
|
2 |
0 gap (28.4ng/ul) |
gDNA HAPMAP C1 (87ng/ul) |
4.47 |
3 |
3.45 |
19.08 |
30
|
3 |
0 gap (28.4ng/ul) |
cDNA |
4.47 |
3 |
20 |
2.53 |
30
|
4 |
20 gap (16.79ng/ul) |
NTC |
7.44 |
3 |
0 |
19.56 |
30
|
5 |
20 gap (16.79ng/ul) |
gDNA HAPMAP C1 (87ng/ul) |
7.44 |
3 |
3.45 |
16.11 |
30
|
6 |
20 gap (16.79ng/ul) |
cDNA |
7.44 |
3 |
20 |
0 |
30.44
|
|
Program
- 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 20h
- To 0 gap probes -> add 3ul AmpLigase enzyme mix (0.5U/ul AmpLigase in 1X AmpLigase buffer)
- To 20 gap probes -> 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.
- No Exo I/III mix was added to NTC samples because lack of Exo I enzyme