07/13/10
- ✓ PCR: flyeGFP, flyCitrine, flyCFP
- ✓ Assemblies: dsOligo flyNLS, PCR flyeGFP, flymCh, flyCitrine, flyCFP into V0120
- ✓ Transformation: transform DH5α Turbo with plasmids for
PCR
- flyeGFP (714 bp)
- flyCFP (711 bp)
- flyCitrine (711 bp)
Reagent |
Volume
|
DNA(plasmid) |
0.4
|
10μM primer mix |
1.0
|
2x GoTaq |
25.0
|
dH2O |
23.6
|
|
50.0 μL
|
BioRad PCR Block A
- 95°C/ 3 min.
- [95°C/ 30 sec.; 57°C/ 10 min.; 72°C/ 10 min.] x 30
- 72°C/ 3 min.
- 4°C/ ∞
> Zymo clean & concentrate
--> Elute w/ 25 μL dH2O
> Proceed to digestion
Assemblies
- KHF01/V0120: ✓ flyNLS/(X/P dsOligo)/21 + (1) V0120/(X/P)/~3200
- KHF02/V0120: (2) flyeGFP/(X/P PCR)/714 + "
- KHF03/V0120: (3) flyCFP/(X/P PCR)/711 + "
- KHF04/V0120: (4) flyCitrine/(X/P PCR)/711 + "
> Digests (Fermentas FD)
Reagent |
Volume |
|
30 μL/lane, 1% agarose
|
DNA (plasmid) |
up to 25 μL
|
10x buffer |
3.0
|
XbaI |
1.0
|
PstI |
1.0
|
dH2O |
---
|
|
30 μL --> 37°C/ ~10 min.
|
> Measure conc.'s
Sample |
OD260 |
260/280 |
ng/μL
|
1. V0120 (X/P) |
0.271 |
2.13 |
13.5
|
2. flyeGFP PCR (X/P) |
1.060 |
1.86 |
53.0
|
2. flyCFP PCR (X/P) |
0.641 |
1.71 |
32.0
|
2. flyCitrine PCR (X/P) |
0.960 |
1.78 |
48.0
|
> Ligations
Ligation |
Plate results (lig : neg crtl) 07/14/10
|
1. flyNLS(X/P dsOligo)/21, 0.3 ng + V0120(X/P)/3200, 25 ng |
KHF01 2 colonies
|
2. flyeGFP(X/P PCR)/714, 11 ng + " |
KHF02 colonies
|
3. flyCFP(X/P PCR)/711, 11 ng + " |
KHF03 5 colonies
|
4. flyCitrine(X/P PCR)/711, 11 ng + " |
KHF04 1 colony
|
2. V0120(X/P)/ 25 ng |
0 colonies
|
|
1 |
2 |
3 |
4 |
5
|
Insert DNA |
1.0 |
1.0 |
1.0 |
1.0 |
---
|
Vector DNA |
2.0 |
2.0 |
2.0 |
2.0 |
2.0
|
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 |
1.0 |
1.0 |
1.0 |
1.0 |
2.0
|
|
10 μL |
10 μL |
10 μL |
10 μL |
10 μL
|
--> R.T./ 10 min.; Add 40 μL DH5α Turbo
--> Results: colonies suspiciously low. Not sure if I mistook EcoRI for XbaI tube when cutting V0120. Do over.
Plasmid Transformation
> Plasmids from the DePace lab
- pJ10:G04380 (flyeGFP+NLS)
- pJ10:G04376 (flyCFP+NLS)
- pJ10:G04382 (flyCitrine+NLS)
- pJ10:G04381 (flymCherry+NLS)
- pAct5C
- dH2O (neg ctrl)
> Transform 0.5 μL (in 10 μL dH2O) into 30 μL DH5α Turbo; plat on 100 μg/mL Amp
|