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

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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
2J06702 X/E Backbone ~2900* ~2900
3F2620 S/P Backbone ~3100* ~3100
4F2620 E/S Front insert 1061 + 2070 1061
5F2620-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:

Plasmid25ul
MM3ul
Enzyme 11ul
Enzyme 21ul
H200ul
Total 30ul

For digest 5, I made a lower reaction volume because it's only to confirm:

Plasmid10ul
MM2ul
Enzyme 11ul
Enzyme 21ul
H206ul
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) 17.3ul19ul
T4 ligase1ul1ul
H200ul0ul
Total 34.6ul38ul

Ligation reactions ran for 15min at room temp. Agitated occasionally.
Transformations

NumberPart(s)ResistanceCell typeul Cells used Method
1 F2620-J06702 Amp DH5aT50ulFast
2 F2620-J06702 Amp DH5aT50ulFast
3TraI (from stef)KanDH5aT30Long
4E0240 m.p 3/3/13 111ng/ul AmpDH5aT30ulFast
5F2620-E0240 m.p. 5/23/13AmpBL2130ulLongish
6K092900 mp 3/3/13 53ng/ulAmpDH5aT30ulFast
7K092900 mp 5/1/13 195.4ng/ul (EL)AmpDH5aT30ulFast
8K092900 mp 4/1/13 12ng/ul (EL)AmpDH5aT30ulFast

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.


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