Hosuk:LabNotes/2014-9-19: Difference between revisions
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>Hosuki78 (Created page with "*LabNote ===ppCOL1A1 test – in NCIH1975=== *To know where the 2nd Rolonies of ppCOL1A1 present in a different cells (NCIH1975) ====Sample==== *S1 : N...") |
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*Add 200ul 1M Tris pH 8.0 at RT for 30min | *Add 200ul 1M Tris pH 8.0 at RT for 30min | ||
*Wash with 1X PBS twice and store in PBS | *Wash with 1X PBS twice and store in PBS | ||
====Result==== | |||
*In Sample1 (has more 1st and 2nd rolonies) --> Most of 2nd rolonies from ppCOL1A1 were found in nuclear. | |||
*In Sample2 (has less 1st and 2nd rolonies) --> Fewer 1st rolonies an 2nd rolonies compare to S1 has, but some of 2nd rolonies from ppCOL1A1 were found outside of nuclear. | |||
[[File:LabnoteImg_Comp_MIP_NCIH1975_ppCOL1A1Test_S1_RCA20140802_1st_2nd_BF_63x_2k_Pos1.png|800px]] | |||
[[File:LabnoteImg_Comp_MIP_NCIH1975_ppCOL1A1Test_S2_RCA20140805_1st_2nd_BF_63x_2k_Pos2.png|800px]] |
Revision as of 22:04, 19 September 2014
ppCOL1A1 test – in NCIH1975
- To know where the 2nd Rolonies of ppCOL1A1 present in a different cells (NCIH1975)
Sample
- S1 : NCIH1975_RCA2014-08-02 : 1st Rolony : good
- S2 : NCIH1975_RCA2014-08-05 : 1st Rolony : less than S1, but not bad
Procedure
- Follow Matt's Ampligase procedure (Matt 2014-09-08)
- Add 100ul Padlock Probe Mix
Components | Volume |
5 U/ul Ampligase | 10 |
10X Ampligase Buffer | 10 |
100uM ppCOL1A1 | 2 |
H2O | 78 |
Total | 100 |
- Incubate at 37C for 30min
- Incubate at 60C for 24hrs
- Wash with 1X PBS twice
- Add 200ul 0.5uM FISSEQ_ppRCA in 2X SSC + 30% formamide (pre-heated to 75C) and incubate at 45C for 30min
- Wash with 2X SSC twice
- Add 200ul RCA Mix and incubate at 30C for 16hrs
- H2O : 172uL
- 10x Buffer : 20uL
- dNTP : 2uL
- a-dUTP : 4uL
- Phi29 (1000 U/µl) : 0.5uL
- Wash with 1X PBS once
- Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) at RT for 1hr
- Wash with 1X PBS twice
- Add 200ul 1M Tris pH 8.0 at RT for 30min
- Wash with 1X PBS twice and store in PBS
Result
- In Sample1 (has more 1st and 2nd rolonies) --> Most of 2nd rolonies from ppCOL1A1 were found in nuclear.
- In Sample2 (has less 1st and 2nd rolonies) --> Fewer 1st rolonies an 2nd rolonies compare to S1 has, but some of 2nd rolonies from ppCOL1A1 were found outside of nuclear.
File:LabnoteImg Comp MIP NCIH1975 ppCOL1A1Test S1 RCA20140802 1st 2nd BF 63x 2k Pos1.png
File:LabnoteImg Comp MIP NCIH1975 ppCOL1A1Test S2 RCA20140805 1st 2nd BF 63x 2k Pos2.png