01/27/11
- ✓ Assemblies: p65 w.t. (PCR); miR sensors
- ✓ Sequencing order: Pc-TF/pSB31 clones; SP1AB mut clones
Assemblies
--> Include set from 01/25/11
- p65 w.t.: p65 w.t./(X/S PCR)/783 + V0120/(X/S dp)/~3200 ✓
- KAH179/176fwd/MV8: KAH179/(E/S)/2941 ✓ + KAH176/MV8/(E/X)/7785✓
- KAH180/176fwd/MV8: KAH180/(E/S)/2743 ✓ + "
- KAH179/177fwd/MV8: KAH179/(E/S)/2941 + KAH177/MV8/(E/X)/7978 ✓
- KAH180/177fwd/MV8: KAH180/(E/S)/2743 + "
- KAH179/178fwd/MV8: KAH179/(E/S)/2941 + KAH178/MV8/(E/X)/7989 ✓
- KAH180/178fwd/MV8: KAH180/(E/S)/2743 + "
> PCR
--> p65 from cDNA templates (Note: p65 up-regulated in stressed cells; try cDNA from rotenone-stressed cells)
- FTRx no rot., 7/08/10
- FTRx + rot., 7/08/10
Reagent |
Volume
|
cDNA (plasmid) |
1.0
|
10 μM primers |
1.0
|
2x GoTaq |
25.0
|
dH2O |
23.0
|
--> BioRad DNA Engine, block A
- 95°C/ 3 min.
- [95°C/ 30 sec; 55°C/ 30 sec; 72 °C/ 1 min.] x30
- 72 °C/ 3 min.
- 4 °C/ ∞
--> Zymo clean PCR; elute w/ 25 μL dH2O
> Digests (Fermentas FD)
Reagent |
Volume |
|
30 μL/lane, 1% agarose Expected = 783 bp Cut out and purify both bands together
|
PCR DNA |
25.0
|
10x buffer |
3.0
|
XbaI |
1.0
|
SpeI |
1.0
|
dH2O |
---
|
|
30 μL --> 37°C/ ~30 min.
|
> Measure conc.'s
Sample |
OD260 |
260/280 |
ng/μL
|
1. p65 w.t. (X/S) |
0.071 |
2.28 |
3.5
|
> Ligations
Ligation |
Plate results (lig : neg crtl) 01/28/11
|
1. p65(X/S)/783, 12 ng + V0120(X/S dp)/~3200, 25 ng |
p65 w.t. 10:1 (Pick 4)
|
2. V0120(X/S dp)/ 25 ng |
|
3. KAH179(E/S)/2941, 26 ng + KAH176fwd/MV8(E/X)/7785, 35 ng |
KAH179/176fwd/MV8 10:1 (Pick 2)
|
4. KAH180(E/S)/2743, 25 ng + " |
KAH180/176fwd/MV8 10:1 (Pick 2)
|
5. KAH176fwd/MV8(E/X)/ 35 ng |
|
6. KAH179(E/S)/2941, 26 ng + KAH177fwd/MV8(E/X)/7978, 35 ng |
KAH179/177fwd/MV8 10:1 (Pick 2)
|
7. KAH180(E/S)/2743, 25 ng + " |
KAH180/177fwd/MV8 10:1 (Pick 2)
|
8. KAH177fwd/MV8(E/X)/ 35 ng |
|
9. KAH179(E/S)/2941, 26 ng + KAH178fwd/MV8(E/X)/7989, 35 ng |
KAH179/178fwd/MV8 10:1 (Pick 2)
|
10. KAH180(E/S)/2743, 25 ng + " |
KAH180/178fwd/MV8 10:1 (Pick 2)
|
11. KAH178fwd/MV8(E/X)/ 35 ng |
|
|
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11
|
Insert DNA |
3.5 |
--- |
3.0 |
4.5 |
--- |
3.0 |
4.5 |
--- |
3.0 |
4.5 |
--- |
|
Vector DNA |
1.0 |
1.0 |
1.7 |
1.7 |
1.7 |
1.6 |
1.6 |
1.6 |
4.2 |
4.2 |
4.2 |
|
2x lgn buf (Roche) |
5.5 |
5.5 |
7.2 |
7.2 |
7.2 |
7.1 |
7.1 |
7.1 |
9.7 |
9.7 |
9.7 |
|
T4 ligase (NEB) |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
|
dH2O |
--- |
3.5 |
1.5 |
--- |
4.5 |
1.5 |
--- |
4.5 |
1.5 |
--- |
4.5 |
|
|
11 μL |
11 μL |
14.4 μL |
14.4 μL |
14.4 μL |
14.2 μL |
14.2 μL |
19.4 μL |
19.4 μL |
19.4 μL |
19.4 μL |
|
--> R.T./ ~15 min.; Add to 50 μL DH5α turbo
Sequencing Order (Genewiz)
--> Results: 1/28/11
- KAH160/pSB31-9, seq_pSB31 fwd: good; insert in fwd orientation (keep this clone)
- KAH160/pSB31-9, seq_pSB31 rev: failed (non-specific)
- KAH165/pSB31-1 (#7), seq_pSB31 fwd: insert in reverse orientation
- KAH165/pSB31-1 (#7), seq_pSB31 rev: failed (ns)
- KAH165/pSB31-4 (#10), seq_pSB31 fwd: good; insert in fwd orientation (keep this clone)
- KAH165/pSB31-4 (#10), seq_pSB31 rev: failed (ns)
- KAH170/pSB31-3 (#2), seq_pSB31 fwd: good; insert in fwd orientation (keep this clone)
- KAH170/pSB31-3 (#2), seq_pSB31 rev: failed (ns)
- KAH170/pSB31-5, seq_pSB31 fwd: insert in reverse orientation
- KAH170/pSB31-5, seq_pSB31 rev: failed (ns)
- SP1AB 2/3mut-1, P0001: discrepancy at bp 230 (A -> G); failed to mutate PstI #1; mutated PstI #2 (keep this clone)
- SP1AB 2/3mut-1, P0002: discrepancy at bp 1141 (A -> G); mutated PstI #3
- SP1AB 2/3mut-2, P0001: discrepancy at bp 230 (A -> G); failed to mutate PstI #1; mutated PstI #2
- SP1AB 2/3mut-2, P0002: discrepancy at bp 1141 (A -> G); mutated PstI #3
- SP1AB 2/3mut-3, P0001: discrepancy at bp 230 (A -> G); failed to mutate PstI #1; mutated PstI #2
- SP1AB 2/3mut-3, P0002: discrepancy at bp 1141 (A -> G); mutated PstI #3
- SP1AB 2/3mut-4, P0001: n/d
- SP1AB 2/3mut-4, P0002: n/d
- SP1AB 2/3mut-5, P0001: n/d
- SP1AB 2/3mut-5, P0002: n/d
- SP1AB 2/3mut-6, P0001: n/d
- SP1AB 2/3mut-6, P0002: n/d
--> Pc-TF/pSB31: Success! Got all three clones for ES cell transfection experiment. Maxi prep and send to Manching. Reverse primer failed (discard/ design new primer).
--> SP1AB mutagenesis: U2OS seems to have two missense mutations in the SP1AB activation domain (bp 230: Gln -> Arg; bp 1141: Ser -> Gly). Keep clone #1 for mutation of PstI #1 (design new longer primer).
|