08/03/10
- ✓ Assemblies: Pc-ATF's and Pc-RFP's + Kozak
- ✓ Minipreps: KFV1 (2)
Minipreps
> Check with separate NotI and SpeI digests
Reagent |
Volume
|
Expected: 1, 3. KFV1/NotI = ~7000(+~35) 2, 4. KFV1/SpeI = ~7000(+~35)
|
15 μL/lane; 1% agarose
|
DNA(plasmid) |
2.0 μL
|
10X buffer |
1.5
|
enzyme |
2.0
|
dH2O |
9.5
|
|
15 μL --> 37°C/ ~15 min.
|
--> Keep clone #1; figure out what primer to use for sequencing confirmation (Ian's Actin 5C sequence data)
Assemblies
- KAH160: Kozak(K0001)/(S/P)/~3200 ✓ + (1) KAH109/(X/P)/1123
- KAH161: " + (2) KAH111/(X/P)/1123
- KAH162: " + (3) KAH112/(X/P)/1123
- KAH163: " + (4) KAH113/(X/P)/2107
- KAH164: " + (5) KAH114/(X/P)/2110
- KAH165: " + (6) KAH148/(X/P)/967
- KAH166: " + (7) KAH150/(X/P)/967
- KAH167: " + (8) KAH151/(X/P)/967
- KAH168: " + (9) KAH152/(X/P)/1931
- KAH169: " + (10) KAH1153/(X/P)/1934
- KAH170: " + (11) KAH115/(X/P)/931
> Digests (Fermentas FD)
Reagent |
Volume |
|
30 μL/lane, 1% agarose
|
DNA (plasmid) |
15.0
|
10x buffer |
3.0
|
XbaI |
1.0
|
PstI |
1.0
|
dH2O |
10.0
|
|
30 μL --> 37°C/ ~30 min.
|
> Measure conc.'s
Sample |
OD260 |
260/280 |
ng/μL
|
1. KAH109 (X/P) |
1.057 |
2.43 |
52.8
|
2. KAH111 (X/P) |
0.827 |
2.21 |
41.4
|
3. KAH112 (X/P) |
1.089 |
2.53 |
54.4
|
4. KAH113 (X/P) |
1.380 |
1.94 |
69.0
|
5. KAH114 (X/P) |
1.022 |
2.20 |
51.1
|
6. KAH148 (X/P) |
0.793 |
2.25 |
39.6
|
7. KAH150 (X/P) |
0.660 |
2.09 |
33.0
|
8. KAH151 (X/P) |
0.631 |
2.08 |
31.6
|
9. KAH152 (X/P) |
1.030 |
2.12 |
51.5
|
10. KAH153 (X/P) |
0.980 |
2.14 |
49.0
|
11. KAH115 (X/P) |
0.628 |
2.21 |
31.4
|
> Ligations
Ligation |
Plate results (lig : neg crtl) 08/04/10
|
1. KAH109(X/P)/1123, 18 ng + K0001(S/P)/3200, 25 ng |
KAH160 >10:1 (Pick 2)
|
2. KAH111(X/P)/1123, 18 ng + " |
KAH161 >10:1 (Pick 2)
|
3. KAH112(X/P)/1123, 18 ng + " |
KAH162 >10:1 (Pick 2)
|
4. KAH113(X/P)/2107, 33 ng + " |
KAH163 >10:1 (Pick 2)
|
5. KAH114(X/P)/2110, 33 ng + " |
KAH164 >10:1 (Pick 2)
|
6. KAH148(X/P)/967, 15 ng + " |
KAH165 >10:1 (Pick 2)
|
7. KAH150(X/P)/967, 15 ng + " |
KAH166 >10:1 (Pick 2)
|
8. KAH151(X/P)/967, 15 ng + " |
KAH167 >10:1 (Pick 2)
|
9. KAH152(X/P)/1931, 30 ng + " |
KAH168 >10:1 (Pick 2)
|
10. KAH153(X/P)/1934, 30 ng + " |
KAH169 >10:1 (Pick 2)
|
11. KAH115(X/P)/931, 15 ng + " |
KAH170 >10:1 (Pick 2)
|
12. K0001(S/P)/ 25 ng |
0 colonies
|
|
1-11 |
12
|
Insert DNA |
1.0 |
---
|
Vector DNA |
0.4 |
0.4
|
2x lgn buf (Roche) |
5.0 |
5.0
|
T4 ligase (NEB) |
1.0 |
1.0
|
dH2O |
2.6 |
2.6
|
|
10μL |
10μL
|
|