12/22/09
- ✓ Cultures: KAH120-125 in MV2 (2 each); KAH135 (2)
- ✓ Digests/ dephos.: Vectors for Strept apt into JDS42 & 43
- ✓ Kinase treatment + oligo annealing: Strept apt
Assemblies
- Strep apt/JDS42: Strept apt/(NotI dsoligo)/50 + (1) JDS42/(NotI)/7468
- Strep apt/JDS43: Strept apt/(NotI dsoligo)/50 + (2) JDS43/(NotI)/7478
> Digests (Fermentas FD)
Reagent |
Volume |
|
30 μL/lane, 1% agarose
|
DNA (plasmid) |
10.0
|
10x buffer |
3.0
|
NotI |
2.0
|
dH2O |
15.0
|
|
30 μL --> 37°C/ ~30 min.
|
> Measure conc.'s
Sample |
OD260 |
260/280 |
ng/μL
|
1. JDS42(NotI) |
0.053 |
2.08 |
5.6
|
2. JDS43(NotI) |
0.139 |
3.24 |
6.9
|
> Dephosphorylation (Roche)
Reagent |
Volume
|
DNA (clean digest) |
up to 17 μL (~100 ng)
|
10x buffer d.p. |
2.0
|
phosphatase |
1.0
|
dH2O |
---
|
|
20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = ~5 ng/μL
|
Kinase treatment & oligo annealing
- Joe's: Strep apt S1 F NotI + Strep apt S1 R NotI
> Kinase treatment
DNA (oligos, 100 μM) |
up to 23 μL (3 μL ea.)
|
10 x PNK (polynucleotide kinase) buffer |
3.0
|
10mM ATP |
2.0
|
T4 polynucleotide kinase (PNK) |
2.0
|
dH2O |
---
|
|
30 μL
|
--> 37°C/ 1.5 hours
--> Add 4 uL 500 mM NaCl
--> 100°C (water bath)/ 5 min.; Cool to R.T. overnight in water bath
|