July 21, 2010
With the cloned out parts and parts from the registry, we began testing digestion/ligation/transformation.
RXN #
|
Digest
|
Cutters
|
Concentrations
|
Volume of template
|
1
|
Chloramphenicol resistant plasmid
|
EcoRI + PstI
|
152.4 ng/uL
|
3.5 uL
|
2
|
Kanamycin resistant plasmid
|
EcoRI + PstI
|
88.6 ng/uL
|
6 uL
|
3
|
Ampicillin resistant plasmid
|
EcoRI + PstI
|
218 ng/uL
|
2.3 uL
|
4
|
M30109
|
EcoRI + SpeI
|
2254.4 ng/uL
|
0.2 uL
|
5
|
R0082
|
XbaI + PstI
|
1125.01 ng/uL
|
0.5 uL
|
6
|
B0034
|
EcoRI + SpeI
|
928 ng/uL
|
0.5 uL
|
7
|
E0033
|
XbaI + PstI
|
1201.5 ng/uL
|
0.42 uL
|
8
|
K091101
|
EcoRI + SpeI
|
2726.1 ng/uL
|
0.2 uL
|
9
|
E0430
|
XbaI + PstI
|
885.98 ng/uL
|
0.58 uL
|
10
|
MicF
|
XbaI + PstI
|
16.78 ng/uL
|
15 uL
|
11
|
MicF
|
EcoRI + PstI
|
48.07 ng/uL
|
10 uL
|
Each one was digested with the following procedure. The restriction enzymes and volume of template of each is on the table above:
- RXN (50 uL)
- 5 uL NEBuffer 2
- 0.5 uL BSA
- 1.0 uL Restriction Enzyme 1
- 1.0 uL Restriction Enzyme 2
- Volume of template from table
- Volume of dH20 that will make the reaction 50 uL overall
After all of the restriction enzyme and other solutions were added to the reactions, they were put in a 37 C bath for 30 minutes. After that, they were put in a 80 C bath for 20 minutes.
Next ligations of all the digestions was done with the following procedure:
Ligations
1
|
2
|
3
|
4
|
5
|
11 uL dH2O
|
11 uL dH2O
|
11 uL dH2O
|
11 uL dH2O
|
11 uL dH2O
|
2.0 uL 10X buffer
|
2.0 uL 10X buffer
|
2.0 uL 10X buffer
|
2.0 uL 10X buffer
|
2.0 uL 10X buffer
|
1.0 uL ligase
|
1.0 uL ligase
|
1.0 uL ligase
|
1.0 uL ligase
|
1.0 uL ligase
|
2.0 uL Digest 2
|
2.0 uL Digest 1
|
2.0 uL Digest 1
|
2.0 uL Digest 1
|
2.0 uL Digest 3
|
2.0 uL Digest 4
|
2.0 uL Digest 6
|
2.0 uL Digest 8
|
2.0 uL Digest 8
|
2.0 uL Digest 11
|
2.0 uL Digest 5
|
2.0 uL Digest 7
|
2.0 uL Digest 9
|
2.0 uL Digest 10
|
Each was incubated for 15 minutes at room temperature (25 C)
Then they were transformed into 50 uL of dH5α cells with 3 uL of ligation solution.
Ligation 1 was inoculated into 200 uL of LB/Kanamycin broth and overnighted in the 37 C room. All the rest of the ligations were inoculated into 200 uL of LB/Chloramphenicol broth and put in the 37 C overnight.
|