Forever Lab
Restriction Digest and Gel Extraction
Gel extracted BL01, BL05, BL09 from V0120 and re-verified colony 1 BL01, BL05, BL09.
| Reagent
|
Volume
|
|
| DNA(BL01, BL05, BL09 respectively) |
20.0 μL
|
| 10X buffer |
3.0 μL
|
| XbaI |
1.0 μL
|
| SpeI |
1.0 μL
|
| dH2O |
5.0 μL
|
| Total |
30 μL --> 37°C/ 30 min.
|
Gel Verification
On the same gel.
| Reagent
|
Volume
|
|
| DNA(BL01, BL05, BL09 respectively) |
5.0 μL
|
| 10X buffer |
1.5 μL
|
| PstI |
1.0 μL
|
| EcorI |
1.0 μL
|
| dH2O |
6.5 μL
|
| Total |
15 μL --> 37°C/ 30 min.
|
Gel exploded so I did not take a picture of it. I had mislabeled BL09 as BL05.
Gel Purification
Cut expected bands from gel. Assumed 200mg which lead to using 600μL ADB during standard gel purification procedure.
| Plasmid |
OD260 |
OD260/280 |
ng/μL
|
| 1. BL01 C2 |
0.008 |
1.55 |
7.94
|
| 1. BL05 C2 |
0.007 |
1.797 |
7.425
|
| 1. BL09 C2 |
0.007 |
1.5 |
7.47
|
Ligation
Calculations resulted in the following:
| |
Ligation |
Negative Control
|
| Insert DNA (BL01, BL05, BL09 respectively) |
12μL |
none
|
| Vector DNA (50 ng) |
1μL |
same
|
| 10x Biolabs T4 Ligase Buffer |
2.0 μl |
same
|
| Biolabs T4 Ligase |
1.0 μl |
same
|
| dH2O |
4.0μL |
16.0μL
|
| |
20.0 μL total |
same
|
|
|
Mix the reaction(s) thoroughly by flicking the tube.
Incubate at room temperature for 30 minutes.
Long Transformation
Using the set up ligation products from above.
- DH5α-T cells were thawed in ice.
- Six 1.5mL tubes were setup and labeled; 1 (BL01), 5 (BL05), 9 (BL09), CMV9 (CMV/MV9, ligation benchmark), rhlI (pos control), Neg (negative control).
- 50μL of thawed cells were transferred into each of these six empty tubes and pipetted up and down to mix.
- The ligation products were then transferred into their respective tubes and flicked to mix. Set back on ice.
- A 1:1000 RhlI plasmid was used as a positive control; 1μL of this plasmid was added to 19μL dH2O to equal the other two products' 20μL total volume. Added to the fifth tube with 50μL thawed cells.
- All tubes incubated on ice for 30 minutes.
- All three tubes were heat shocked on the heat block at 42°C for 30 seconds and then placed back on ice for 2 minutes.
- 900μL LB broth (no antibiotic) was pipetted into each of the three tubes.
- Tubes were taped into an empty plate. The plate was taped to the shaker within the incubator.
- Incubated on shaker for 1 hour at 37°C and 240rpm.
Did NOT:
- Tubes centrifuged for 2 minutes at 6000 x g.
- 600μL supernatant discarded from each tube.
- Vortexed tubes to re-suspend cells.
DID:
- 300μL of re-suspended cells were then plates on six marked plates and placed in the incubator for overnight growth.
|
