07/14/10
- ✓ Cultures: pJ10's and pAct5C plasmid transformations (2 mL ~5 hr. Turbo cultures)
- ✓ Assemblies: re-try cloning from yesterday (may have incorrectly cut V0120)
Minipreps
> Check pJ10's with BamHI/KpnI digests
> Check pAct5C with BioBrick enzymes:
- EcoRI/ XbaI
- NotI/ XbaI
- SpeI/ XbaI
- PsI/ XbaI
- XbaI
- 0 enzyme
Reagent |
Volume
|
Expected: 1. pJ10:G04380(eGFP) = 760, 4179 2. pJ10:G04376(CFP) = 757, 4179 3. pJ10:G04382(Citrine) = 757, 4179 4. pJ10:G04381(mCh) = 751, 4179 5. pAct5C E/X = ? 6. pAct5C N/X = linear (full length) 7. pAct5C S/X = 2 bands (luciferase, vector) 8. pAct5C P/X = ? 9. pAct5C X = linear (full length) 10. pAct5C ctrl = supercoiled
|
15 μL/lane; 1% agarose
|
DNA(plasmid) |
2.0
|
10X buffer |
1.5
|
enzyme 1 |
1.0
|
enzyme 2 |
1.0
|
dH2O |
9.5
|
|
15 μL --> 37°C/ ~15 min.
|
--> Conclusions: pAct5C contains EcoRI and PstI, but not sure where since there is no sequence data. Will cut out renilla luc w/ SpeI/ NotI and replace with mini BB cloning site (NotI-SpeI). Order primers tomorrow.
Assemblies
> See 07/13/2010
> Digests (Fermentas FD)
--> Re-digest V0120
Reagent |
Volume |
|
|
DNA (plasmid) |
25.0
|
10x buffer |
3.0
|
XbaI |
1.0
|
PstI |
1.0
|
dH2O |
---
|
|
30 μL --> 37°C/ ~15 min.
|
> Measure conc.'s
Sample |
OD260 |
260/280 |
ng/μL
|
1. V0120 (X/P) |
0.642 |
3.48 |
32.1
|
> Ligations
Ligation |
Plate results (lig : neg crtl) 07/15/10
|
1. flyNLS(X/P dsOligo)/21, 0.4 ng + V0120(X/P)/3200, 30 ng |
flyNLS 6 colonies (Pick 2)
|
2. flyeGFP(X/P PCR)/714, 13 ng + " |
flyeGFP 13 colonies (Pick 2)
|
3. flyCFP(X/P PCR)/711, 13 ng + " |
flyCFP 9 colonies (Pick 2)
|
4. flyCitrine(X/P PCR)/711, 13 ng + " |
flyCitrine 6 colony (Pick 2)
|
2. V0120(X/P)/ 25 ng |
1 colony
|
|
1 |
2 |
3 |
4 |
5
|
Insert DNA |
1.0 |
1.0 |
1.0 |
1.0 |
---
|
Vector DNA |
1.0 |
1.0 |
1.0 |
1.0 |
1.0
|
2x lgn buf (Roche) |
5.0 |
5.0 |
5.0 |
5.0 |
5.0
|
T4 ligase (NEB) |
1.0 |
1.0 |
1.0 |
1.0 |
1.0
|
dH2O |
2.0 |
2.0 |
2.0 |
2.0 |
3.0
|
|
10 μL |
10 μL |
10 μL |
10 μL |
10 μL
|
--> R.T./ 10 min.; Add 40 μL DH5α Turbo
|