Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2013/05/23

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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.