Page Size Selection of 0 gap probes[edit]
USER/DpnII digested 0 gap probes:
Noi made 11.36ug/~400ul 2013-5-29
I made 10.58ug/180ul 2013-5-16
To run ~3ug/gel, I use ~100ul of probes that Noi made for each of the 4 gels
- Ran TBU gel at 220V for 30 min with samples and ladder prepared below
- Heated TBE buffer in microwave
- Denatured samples/ladder for 10 min at 75C before putting on cold rack for 1 min
- Size selected for brightest band below 200bp
Components
|
1X Volume
|
4X Volume
|
Enzyme digested probes (~11.36/400ul) |
106 |
424
|
TBE-Urea Buffer 2X |
106 |
424
|
H2O |
0 |
0
|
Total |
212 |
848
|
|
Components
|
1X Volume
|
4X Volume
|
Low MW DNA ladder (1 ug/ul) |
1 |
4
|
TBE-Urea Buffer 2X |
5 |
20
|
H2O |
4 |
16
|
total |
10 |
40
|
|
File:2013-06-26 Agi26k 0gap SizeSelect1.jpg
File:2013-06-26 Agi26k 0gap SizeSelect2.jpg
File:2013-06-26 Agi26k 0gap SizeSelect3.jpg
File:2013-06-26 Agi26k 0gap SizeSelect4.jpg
- Put cut out gels in 0.5mL tube with holes at bottom in 2 mL tubes (6 tubes for 4 gels)
- Centrifuged the tubes at 15,000 rpm, for 3 min at RT.
- Transfered the gel remaining in 0.5 mL tube to 2 mL tube below with pipette tip.
- Added 675 ul of 1X TE buffer (because 1.5x more gel compared to normal protocol using 1.5mL tube)
- Vortexed for 45-60 min at 37 C in incubator
- Centrifuged at 15,000 rpm for 3 min at RT
- Transfered the clear spnt. to Nanosep column and centrifuged at 15,000 rpm for 3 min
- Transfered the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min
- Transfered spnt to fresh 1.5 mL tube (volume ~ 500ul/ tube)
- Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 40ul of 3M NaoAc pH 5.2
- Vortexed and placed the 8 tubes at -80C overnight (had to use 8 because volume was too much for 6)
Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-6-27
First Strand cDNA synthesis[edit]
- Followed NEB E6300S protocol: [1]
Got 2ul aliquot of Human Brain Reference RNA from Blue at concentration 1ug/ul
- Mixed components in two 0.2 ml microfuge tubes labelled HBBR 1ug cDNA
Components
|
Volume
|
2X Volume
|
RNA |
1 ul (1 ug) |
2 ul (2 ug)
|
d(T)23VN (50uM) |
2 ul |
4 ul
|
H2O |
5 ul |
10 ul
|
Total |
8 ul |
16 ul
|
|
- Denatured RNA for 5 min at 70C and then put on ice
- Tightened the thermocycler too much and bent the tube slightly out of shape
- Added the following to each 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
- Kept on ice
Second Strand cDNA synthesis[edit]
- Follow NEB E6111S protocol: [2]
- Added 48 ul H20 to each tube
- 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)
- Measured DNA conc with Nandrop:
- 43ul at 63.7ng/ul (2.74 ug) sample left in each tube
- "High yield" because RNA was not digested and went from single stranded to double stranded; slightly higher but consistent with last time 2013-3-9