Entry title
Miniprepped colonies picked yesterday
Part number |
260 |
280 |
260/280 |
ng/ul
|
F2620 miniprep 1 |
0.052 |
0.027 |
1.881 |
51.662
|
F2620 miniprep 2 |
0.024 |
0.014 |
1.679 |
23.941
|
J06702 miniprep 1 |
0.039 |
0.022 |
1.766 |
38.85
|
J06702 miniprep 2 |
0.057 |
0.032 |
1.782 |
56.501
|
F2620-E0240 miniprep |
0.046 |
0.026 |
1.794 |
45.856
|
Want to build F2620-J06702. Will try having F2620 as a front insert and J06702 as the backbone as well as F2620 as the backbone and J06702 as the rear insert.
Digests:
Number |
Part number |
Enzymes |
Insert or Bb |
Expected band sizes |
Extracted band
|
1 |
J06702 |
X/P |
Rear insert |
869 + 2070 |
869
|
2 |
J06702 |
X/E |
Backbone |
~2900* |
~2900
|
3 |
F2620 |
S/P |
Backbone |
~3100* |
~3100
|
4 |
F2620 |
E/S |
Front insert |
1061 + 2070 |
1061
|
5 |
F2620-E0240 |
X/P |
N/A |
1920 + 2070 |
N/A
|
'*Not totally sure what the backbone length is.
Because of the low mini prep yields for 1-4, I used more DNA for digests 1-4:
Plasmid |
25ul
|
MM |
3ul
|
Enzyme 1 |
1ul
|
Enzyme 2 |
1ul
|
H20 |
0ul
|
Total |
30ul
|
For digest 5, I made a lower reaction volume because it's only to confirm:
Plasmid |
10ul
|
MM |
2ul
|
Enzyme 1 |
1ul
|
Enzyme 2 |
1ul
|
H20 |
6ul
|
Total |
20ul
|
Digests ran for 20 min at 37deg
Gel looked good but the J06702 vector is ~1kb longer than expected.
Part number |
260 |
280 |
260/280 |
ng/ul
|
J06702 X/P |
0.001 |
0.001 |
0.833 |
0.526
|
J06702 X/E |
0.01 |
0.007 |
1.446 |
10.002
|
F2620 S/P |
0.015 |
0.009 |
1.709 |
15.477
|
F2620 E/S |
0.002 |
0.001 |
1.8 |
1.915
|
Ligations
|
Rxn 1 |
Rxn 2
|
Bb |
F2620 S/P 3.3ul |
J06702 X/E 5ul
|
Insert |
J06702 X/P 13ul |
F2620 E/S 13ul
|
2x Roche buffer (Behzad) |
17.3ul |
19ul
|
T4 ligase |
1ul |
1ul
|
H20 |
0ul |
0ul
|
Total |
34.6ul |
38ul
|
Ligation reactions ran for 15min at room temp. Agitated occasionally.
Transformations
Number |
Part(s) |
Resistance |
Cell type |
ul Cells used |
Method
|
1 |
F2620-J06702 |
Amp |
DH5aT |
50ul |
Fast
|
2 |
F2620-J06702 |
Amp |
DH5aT |
50ul |
Fast
|
3 |
TraI (from stef) |
Kan |
DH5aT |
30 |
Long
|
4 |
E0240 m.p 3/3/13 111ng/ul |
Amp |
DH5aT |
30ul |
Fast
|
5 |
F2620-E0240 m.p. 5/23/13 |
Amp |
BL21 |
30ul |
Longish
|
6 |
K092900 mp 3/3/13 53ng/ul |
Amp |
DH5aT |
30ul |
Fast
|
7 |
K092900 mp 5/1/13 195.4ng/ul (EL) |
Amp |
DH5aT |
30ul |
Fast
|
8 |
K092900 mp 4/1/13 12ng/ul (EL) |
Amp |
DH5aT |
30ul |
Fast
|
For 3 and 5, iced for 5min, heat shock for 45s at 42deg, iced for 2 min. Plated 5, shook 3 with 800ul SOC for 30min then plated.
|