Summary
- Gel Extracted/Purified BL01
- Retransformed BL01 & BL05
- Digested CMV/MV9 with XbaI for LCR
- LCR
- Digested CMV/MV9 with XbaI and desphos'd, clean and concentrated for long transformation
- Long Transformation
Gel Extraction/Purification of BL01
Reagent
|
Volume
|
Expected: 1. BL01 = 2500bp
|
DNA(BL01) |
15.0 μL
|
10X buffer |
3.0 μL
|
XbaI |
1.0 μL
|
SpeI |
1.0 μL
|
dH2O |
10 μL
|
Total |
30 μL --> 37°C/ 15 min.
|
Gel Purification: BL01/V0120
- Using Prior Digest
- Placed 15μL 1kB Ladder in well one and 30μL of BL01 digest in well two.
- Ran gel at 110V for 55 minutes.
- Placed gel on UV transilluminator and cut out desired band(2500bp).
- Used Zymo gel purification kit.
- Assumed 200mg gel, added 600μL ADB to the 1.5mL tube containing extracted gel piece.
- Put on heat block at 55°C for 8 minutes.
- Put ~750μL gel/ADB into spin column, centrifuged at max rpm for 30 seconds.
- Pipetted 200μL wash buffer to the tube, centrifuged at max rpm for 30 seconds. Repeated once.
- Transferred column to new labeled 1.5mL tube.
- Pipetted 15μL elution buffer to the column.
- Spun at max rpm for 30 seconds.
Concentration
Plasmid |
OD260 |
OD260/280 |
ng/μL
|
1. Purified BL01 |
0.03 |
2.05 |
30.272
|
Digest and Dephos of CMV/MV9 with XbaI
DNA(CMV/MV9) Concentration: 163ng/μL |
5.0 μL
|
10X buffer |
1.5 μL
|
XbaI |
1.0 μL
|
dH2O |
7.5 μL
|
Total |
15 μL --> 37°C/ 15 min.
|
- Clean and concentrated XbaI cut CM9. Eluted with 10μL dH2O.
Mixture from Above Digest |
10.0 μL
|
10X Phosphotase buffer |
2.0 μL
|
Phosphotase |
1.0 μL
|
dH2O |
7.0 μL
|
Total |
20 μL --> 37°C/ 10 min. 75°C for 2 min.
|
- Clean and concentrated DNA after dephos step; eluted with 12μL dH2O.
Plasmid |
OD260 |
OD260/280 |
ng/μL
|
1. Purified DNA |
0.017 |
1.953 |
17.425
|
LCR
LCR Calculations
- DNA Part to Oligo Bridge is 1:10 (DNA part ratio is 1:1)
- X ng insert = (bp inset / bp vector) x 1 x 50 ng vector
- X ng BL01&9 = (2520bp/5197bp)*1*50ng vector = 24.24ng BL01&9
- Volume BL01&9 = 24.24ng*(1μL/10ng) = 2.424μL
- Volume CMV/MV9 = 50ng*(1μL/33ng) = 1.28μL
- 2:1 vector to insert use 2.56μL vector.
|
BL01 |
BL05
|
Insert DNA |
2.5μL |
2.5μL
|
XbaI Cut and Cleaned Vector DNA (CMV/MV9) |
1.3μL |
2.6μL
|
Oligo Bridge1 |
2.5μL |
2.5μL
|
Olido Bridge2 |
2.5μL |
2.5μL
|
10X Ampligase Buffer |
2.5 μl |
2.5μL
|
Ampligase |
0.5μl |
0.5μL
|
Betaine |
0μL |
0μL
|
DMSO |
0μL |
0μL
|
dH2O |
13.2μL |
11.9μL
|
Total |
25.0 μL |
25μL
|
Mix the reaction(s) thoroughly by flicking the tube. Placed in thermocycler on LCR setting.
|
Normal Transformation (BL01 & BL05 in V0120)
- Warmed selection agar plates at 37°C.
- Incubated DH5α Turbo competent cells on ice just until thawed. Use 50 μL per transformation.
- Added 50 μL thawed cells to 5μL LCR product. Immediately placed on ice and incubated for 10 min. (Do not heat shock; No 30 min. recovery is required for Amp resistance)
- Pipetted the total volume of cells + LCR reaction onto the agar; spread using sterile glass beads.
- Incubated overnight at 37°C.
Long Transformation
Using the set up ligation products from above.
- DH5α-T cells were thawed in ice.
- Two 1.5mL tubes were setup and labeled; LCR BL01 & Long Transformation BL01.
- 60μL of thawed cells were mixed and then transferred into each of these two empty tubes.
- The ligation products were then transferred into their respective tubes and flicked to mix. Set back on ice.
- All tubes incubated on ice for 35 minutes.
- All tubes were heat shocked on the heat block at 42°C for 30 seconds and then placed back on ice for 2 minutes.
- 900μL SOC Medium 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.
- Centrifuged for 1.5 minutes at 9 x g.
- 500μL media removed from each tube.
- Resuspended cell pellet in remaining 400μL media.
- 300μL cells transferred onto respective plate.
- Sterile glass beads used to spread cells onto plate.
- Placed in the incubator for overnight growth.
|