Matt:LabNotes/2013-6-26: Difference between revisions
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>Mzcai |
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===Page Size Selection of 0 gap probes=== | ===Page Size Selection of 0 gap probes=== | ||
USER/DpnII digested 0 gap probes: | USER/DpnII digested 0 gap probes: | ||
Noi made 11.36ug/~400ul [http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-5-29] | Noi made 11.36ug/~400ul [http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-5-29 2013-5-29 ] | ||
I made 10.58ug/180ul [http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-5-16] | I made 10.58ug/180ul [http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-5-16 2013-5-16 ] | ||
<!--If Combined: 11.36ug + 10.58ug / (410ul + 180ul) = 21.94ug/590ul = 37.19ng/ul (590ul)--> | <!--If Combined: 11.36ug + 10.58ug / (410ul + 180ul) = 21.94ug/590ul = 37.19ng/ul (590ul)--> | ||
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| align="center" style="background:#f0f0f0;"|'''4X Volume''' | | align="center" style="background:#f0f0f0;"|'''4X Volume''' | ||
|- | |- | ||
| | | Low MW DNA ladder (1 ug/ul)||1||4 | ||
|- | |- | ||
| TBE-Urea Buffer 2X||5||20 | | TBE-Urea Buffer 2X||5||20 | ||
Line 43: | Line 43: | ||
|} | |} | ||
[[File:2013-06-26_Agi26k_0gap_SizeSelect1.jpg | 450px]] | |||
[[File:2013-06-26_Agi26k_0gap_SizeSelect2.jpg | 450px]] | |||
[[File:2013-06-26_Agi26k_0gap_SizeSelect3.jpg | 450px]] | |||
[[File:2013-06-26_Agi26k_0gap_SizeSelect4.jpg | 450px]] | |||
*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==== | ====First Strand cDNA synthesis==== | ||
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*Measured DNA conc with Nandrop: | *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 [http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-3-9 2013-3-9] |
Latest revision as of 16:46, 27 June 2013
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