01/31/11
- ✓ Miniprep digests: p65 w.t.(4), KAH179/176, 180/176, 179/177, 180/177, 179/178, 179/178/MV8 (2 each)
Minipreps
> p65 w.t.: X/P
> Others: E/ApaLI (Note: KAH176, 177, 178 have 2 ApaLI mCherry sites)
| Reagent |
Volume
|
Expected:
1-4. p65 w.t. = 3200, 464, 313, 6
MV8 = 1694, 1243, 1135, 288
Inserts + 234 in last fragment...
5,6. 179/176/MV8 = 4550, 711, 1158
7,8. 180/176/MV8 = 4352, 711, 1158
9,10. 179/177/MV8 = 4550, 711, 1351
11,12. 180/177/MV8 = 4352, 711, 1351
13,14. 179/178/MV8 = 4550, 711, 1362
15,16. 180/178/MV8 = 4352, 711, 1362
|
15 μL/lane; 1% agarose
|
| DNA(plasmid) |
2.0 μL
|
| 10X buffer |
1.5
|
| enzyme 1 |
1.0
|
| enzyme 2 |
1.0
|
| dH2O |
9.5
|
| |
15 μL --> 37°C/ ~15 min.
|
--> Conclusions:
- p65 cloning worked
- Unsure about miR sensor assemblies. Largest band shorter than expected and there are many additional bands. Will check restriction sites and send circled clones off for sequencing.
---> mCherry has ApaLI site. Recalculated expected fragments. All make sense except for ~2 kb bands, which are probably partial digests (711+last fragment). Test these by transfection into U2OS.
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Project name
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