05/28/15
- David Tze - assemblies: linkers + hPCD
- Ben - DBN007 5 mL cultures
David Tze - assemblies: linkers + hPCD
- DT001_pUC57: hPCD(E/S)/186 + PLflex_pUC57(E/X)/2763
- DT002_pUC57: hPCD(E/S)/186 + PLflex4_pUC57(E/X)/2930
- DT003_pUC57: hPCD(E/S)/186 + PLrigid_pUC57(E/X)/2763
- DT004_pUC57: hPCD(E/S)/186 + PLrigid4_pUC57(E/X)/2931
Reagent
|
Rxn1
|
Rxn2-5
|
Expected bands: 1. hPCD(E/S) = 3200, 186* 2. PLflex_pUC57(E/X) = 2763* 3. PLflex4_pUC57(E/X) = 2930* 4. PLrigid_pUC57(E/X) = 2763* 5. PLrigid4_pUC57(E/X) = 2931*
(*) Bands that were cut out for purification
|
30 μL/lane, 1% agarose; Ladder
|
DNA (plasmid) |
25.0 |
15.0
|
10x buffer |
3.0 |
3.0
|
EcoRI |
1.0 |
1.0
|
enzyme 2 |
1.0 |
1.0
|
dH2O |
--- |
10.0
|
|
30 μL
|
--> 37°C/ ~30 min.
- Gel purification
- Sigma GenElute Gel purification kit
- Elute & back-elute w/ 25 μL elution sln.
Sample |
OD260 |
260/280 |
ng/μL
|
1. hPCD(E/S) |
--- |
--- |
---
|
2. PLflex_pUC57(E/X) |
--- |
--- |
---
|
3. PLflex4_pUC57(E/X) |
--- |
--- |
---
|
4. PLrigid_pUC57(E/X) |
--- |
--- |
---
|
5. PLrigid4_pUC57(E/X) |
--- |
--- |
---
|
- Readings are very odd, which is typical for the Sigma gel extraction products.
- Will try my luck with these and use an arbitrary volume of 2.0 insert and 2.0 vector.
- Good insert : vector ratio should not be difficult to achieve since insert is very short.
- Ligations
- 2:1 ratio calculation: 186 bp insert / ~2800 bp vector * 2 * 50 = 6.64 ng insert
- DT001_pUC57: hPCD(E/S)/186 + PLflex_pUC57(E/X)/2763
- DT002_pUC57: hPCD(E/S)/186 + PLflex4_pUC57(E/X)/2930
- DT003_pUC57: hPCD(E/S)/186 + PLrigid_pUC57(E/X)/2763
- DT004_pUC57: hPCD(E/S)/186 + PLrigid4_pUC57(E/X)/2931
- PLflex_pUC57(E/X)/2763
- Note: not enough plates to do neg. ctrls for all vectors. Used just one of the vectors
Reagent
|
Rxn1-4
|
Rxn5
|
Insert DNA |
2.0 |
---
|
Vector DNA |
2.0 |
2.0
|
2x lgn buf (Roche) |
5.0 |
5.0
|
T4 ligase (NEB) |
1.0 |
1.0
|
dH2O |
--- |
2.0
|
|
10.0 μL |
10.0 μL
|
RESULTS
- Looks successful - David Tze: ~200 colonies on the ligation plates, 1 colony on the neg. ctrl plate.
- David Tze: picked two colonies from each plate (1-4) for streak library and 5 mL cultures
Ben - DBN007 5 mL cultures
- Minipreps
- Sigma GenElute kit, elute w/ 75 μL elution sln.
Reagent
|
Volume
|
Expected: 1-2. DBN007_pSB1A3 = 2197, 2132 DBN001 = 2132, 224
|
15 μL/lane; 1% agarose; Ladder
|
DNA(plasmid) |
2.0 μL
|
10X buffer |
1.5
|
EcoRI |
1.0
|
SpeI |
1.0
|
dH2O |
9.5
|
|
15 μL
|
--> 37°C/ ~15 min.
CONCLUSIONS
- Looks successful. 2197, 2132 bands are hard to resolve at the 2000 kb range.
- Submit the plasmids for sequencing
|