The printable version is no longer supported and may have rendering errors. Please update your browser bookmarks and please use the default browser print function instead.
Karmella's BioBrick Cloning
|
<html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>
|
04/14/13
- New vectors: design new vectors for mammalian expression
- Order oligos
New mammalian expression vectors MV9 and MV10
- Use the Oligator tool to design oligo-assembly inserts for "MV6" (pcDNA3.1+ puro, mammalian transfection vector; CMV promoter)
- MV9 - will carry Kozak-XbaI-NLS-6His-stop (Brady's and Behzad's project)
- MV10 - will carry Kozak-XbaI-stop
- Oligo assembly method will be used to make double stranded DNA with SpeI overhangs
- Inserts will be ligated to XbaI cut MV6 to destroy the preexisting XbaI site
- Clones will be screened for proper orientation and insert copy number
Oligator results:
Kozak-XbaI-NLS-6His-stop
CTAGTCCCGCCGCCACCATGGAGTCTAGACCCAAGAAAAAGCGCAAGGTACACCATCACCACCATCACGCGTAAAGCTGAGA
AGGGCGGCGGTGGTACCTCAGATCTGGGTTCTTTTTCGCGTTCCATGTGGTAGTGGTGGTAGTGCGCATTTCGACTCTGATC
Assemblies
- BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
- BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
Reagent
|
Volume
|
|
DNA (plasmid) |
up to 25 μL
|
10x buffer |
3.0
|
enzyme 1 |
1.0
|
enzyme 2 |
1.0
|
dH2O |
---
|
|
30 μL --> 37°C/ ~30 min.
|
Sample |
OD260 |
260/280 |
ng/μL
|
1. Digested part (a/b) |
--- |
--- |
---
|
2. Digested part (c/d) |
--- |
--- |
---
|
- Dephosphorylation (Roche)
Reagent
|
Volume
|
DNA (clean digest) |
up to 17 μL (500 ng)
|
10x buffer d.p. |
2.0
|
phosphatase |
1.0
|
dH2O |
---
|
|
20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
|
Ligation |
Plate results (lig : neg crtl) mm/dd/yy
|
1. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng |
new BioBrick #:1 (Pick #)
|
2. vector(c/d)/ ## ng |
|
|
1 |
2 |
|
Insert DNA |
### |
--- |
|
Vector DNA |
### |
### |
|
2x lgn buf (Roche) |
### |
### |
|
T4 ligase (NEB) |
1.0 |
1.0 |
|
dH2O |
### |
### |
|
|
# μL |
# μL |
|
Oligo annealing
- New BB 1
- New BB 2
DNA (oligos, 100 μM) |
up to 18 μL (3 μL ea.)
|
10x annealing buffer |
2.0
|
dH2O |
---
|
|
20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight
|
|