Matt:LabNotes/2013-7-9
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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 |