Summary
- LCR
- Met Kodibagkar grad student Shubanghi
LCR
Oligo bridges initially 30nm pellet.
Diluted down to 30nM in 25μL reaction volume (Added 300μL H2O to IDT tube with pelleted DNA to yield 100μM, added 3μL of that dilution to 97μL H2O to yield 3μM, added 5μL of that dilution to 45μL H2O to yield 300nM, added 2.5μL of this dilution to the 25μL reaction volume for LCR).
Dephos XbaI linearized MV10 gel purification product
XbaI gel purified MV10 |
5.0 μL
|
10X Phosphotase buffer |
2.0 μL
|
Phosphotase |
1.0 μL
|
dH2O |
12.0 μL
|
Total |
20 μL --> 37°C/ 10 min. 75°C for 2 min.
|
LCR Calculations
- 1:1 vector to insert
- BL01: (2520bp/5197bp)(1)(50ng)=24.24ng
- 24.24ng(1μL/11.3ng)=2.145μL XbaI/SpeI cut BL05 Sample2
- BL05: (2520bp/5197bp)(1)(50ng)=24.24ng
- 24.24ng(1μL/18.5ng)=1.31μL XbaI/SpeI cut BL05 Sample1
- 50ngCMV/MV9(1μL/59ng)=0.847μL XbaI cut dephos'd CMV/MV9
- 50ngCMV/MV9(1μL/15ng)=3.33μL XbaI cut dephos'd CMV/MV9
|
BL05.1 |
BL05.2 |
Neg
|
Insert DNA |
1.3μL |
2.2μL |
0μL
|
XbaI Cut Dephos'd CMV/MV9 |
1μL |
3.33μL |
0μL
|
Oligo Bridge1 |
2.5μL |
2.5μL |
2.5μL
|
Olido Bridge2 |
2.5μL |
2.5μL |
2.5μL
|
10X Ampligase Buffer |
2.5 μl |
2.5μL |
2.5μL
|
Ampligase |
1μl |
1μL |
1μL
|
Betaine |
0μL |
0μL |
0μL
|
DMSO |
0μL |
0μL |
0μL
|
dH2O |
14.2μL |
11μL |
14.5μL
|
Total |
25.0 μL |
25μL
|
Mix the reaction(s) thoroughly by flicking the tube. Placed in thermocycler on LCR setting. *Volume reduced to ~10μL after removal from PCR machine. Settings incorrect?
|
Long Transformation
Using the set up ligation products from above.
- Warmed selection agar plates at 37°C.
- DH5α-T cells were thawed in ice.
- 1.5mL tubes were setup and labeled.
- 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 at 37°C.
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