Matt:LabNotes/2014-3-19: Difference between revisions
Jump to navigation
Jump to search
>Mzcai (Created page with "==Padlock Probe Concentration Test using Fixed Template== *96-well plate NCI-H1975 Cells **Alan already marked locations with good cell density *Test concentrations NegCtrl, ...") |
>Mzcai mNo edit summary |
||
Line 1: | Line 1: | ||
==Padlock Probe Concentration Test using Fixed Template== | ==Padlock Probe Concentration Test using Fixed Template== | ||
*96-well plate NCI-H1975 Cells | *96-well plate NCI-H1975 Cells (C8, C9, C10, D8, D9, D10, E8, E9, E10, F9) | ||
**Alan already marked locations with good cell density | **Alan already marked locations with good cell density | ||
*Test concentrations NegCtrl, 10pM, 50pM, 100pM, 10nM | *Test concentrations NegCtrl-0pM, 10pM, 50pM, 100pM, 10nM | ||
**2 wells for each | **2 wells for each | ||
===Fixing Template in cells=== | ===Fixing Template in cells=== | ||
Line 17: | Line 16: | ||
#* Incubate @ RT for 30min | #* Incubate @ RT for 30min | ||
#Wash with 1X PBS twice | #Wash with 1X PBS twice | ||
===Anneal Padlock Probes=== | |||
*1X Ampligase Buffer | |||
*0.5 U/ul Ampligase | |||
*Incubate @ 45C for 24 hours (Started at 4pm) | |||
**Wrapped in parafilm and put in ziploc with wet towel | |||
====Negative Control 0pM (C8 & C9)==== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Component''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume''' | |||
|- | |||
| 10X Ampligase Buffer||10 | |||
|- | |||
| 5U/ul Ampligase||10 | |||
|- | |||
| H2O||80 | |||
|- | |||
| Total||100 | |||
|} | |||
====10pM (D8 & D9)==== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Component''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume''' | |||
|- | |||
| 10X Ampligase Buffer||10 | |||
|- | |||
| 1nM ppMALAT1||1 | |||
|- | |||
| 5U/ul Ampligase||10 | |||
|- | |||
| H2O||79 | |||
|- | |||
| Total||100 | |||
|} | |||
====50pM (E8 & E9)==== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Component''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume''' | |||
|- | |||
| 10X Ampligase Buffer||10 | |||
|- | |||
| 1nM ppMALAT1||5 | |||
|- | |||
| 5U/ul Ampligase||10 | |||
|- | |||
| H2O||75 | |||
|- | |||
| Total||100 | |||
|} | |||
====100pM (C10 & D10)==== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Component''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume''' | |||
|- | |||
| 10X Ampligase Buffer||10 | |||
|- | |||
| 1nM ppMALAT1||10 | |||
|- | |||
| 5U/ul Ampligase||10 | |||
|- | |||
| H2O||70 | |||
|- | |||
| Total||100 | |||
|} | |||
====10nM (E10 & F9)==== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Component''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume''' | |||
|- | |||
| 10X Ampligase Buffer||10 | |||
|- | |||
| 1uM ppMALAT1||1 | |||
|- | |||
| 5U/ul Ampligase||10 | |||
|- | |||
| H2O||79 | |||
|- | |||
| Total||100 | |||
|} |
Revision as of 22:19, 19 March 2014
Padlock Probe Concentration Test using Fixed Template
- 96-well plate NCI-H1975 Cells (C8, C9, C10, D8, D9, D10, E8, E9, E10, F9)
- Alan already marked locations with good cell density
- Test concentrations NegCtrl-0pM, 10pM, 50pM, 100pM, 10nM
- 2 wells for each
Fixing Template in cells
- Pre-heat 10uM MALAT1_Template in 2X SSC to 85C (50ul 100uM MALAT1_Template + 50ul 20X SSC + 400ul H2O)
- Add 50ul to each well
- Incubate @RT for 15min
- Aspirate but don't wash
- Add 50ul BS(PEG)9 mix (10ul BS(PEG)9 + 490ul 1X PBS)
- Incubate @RT for 1hr
- Wash with 1X PBS twice
- Add 50ul 1M Tris pH 8.0
- Incubate @ RT for 30min
- Wash with 1X PBS twice
Anneal Padlock Probes
- 1X Ampligase Buffer
- 0.5 U/ul Ampligase
- Incubate @ 45C for 24 hours (Started at 4pm)
- Wrapped in parafilm and put in ziploc with wet towel
Negative Control 0pM (C8 & C9)
Component | Volume |
10X Ampligase Buffer | 10 |
5U/ul Ampligase | 10 |
H2O | 80 |
Total | 100 |
10pM (D8 & D9)
Component | Volume |
10X Ampligase Buffer | 10 |
1nM ppMALAT1 | 1 |
5U/ul Ampligase | 10 |
H2O | 79 |
Total | 100 |
50pM (E8 & E9)
Component | Volume |
10X Ampligase Buffer | 10 |
1nM ppMALAT1 | 5 |
5U/ul Ampligase | 10 |
H2O | 75 |
Total | 100 |
100pM (C10 & D10)
Component | Volume |
10X Ampligase Buffer | 10 |
1nM ppMALAT1 | 10 |
5U/ul Ampligase | 10 |
H2O | 70 |
Total | 100 |
10nM (E10 & F9)
Component | Volume |
10X Ampligase Buffer | 10 |
1uM ppMALAT1 | 1 |
5U/ul Ampligase | 10 |
H2O | 79 |
Total | 100 |