Berk2006-ConjugationTeam
JCA_06/17/06
We had some difficulty with the traJF and traJR stocks for our initial tests of conjugation. It is unclear what plasmid the -80 stocks are inserted into. We tried to grow up the "J01024" from Box2>7v4 in carbencillin media. It didn't grow. Yesterday we grew up the "J01024/KAN" in Box1>9v1. It grew just fine and the cell pellet was slightly pink due to its Ptet-RFP cassette. It is unclear whether this is pSB1AK3 or perhaps a pSB*K* plasmid? Unclear, but we're going to transfer it into pSB3C6 anyway, so who cares. In addition, I was concerned that the oriTF plasmids might have CmR markers on them. The biobricks annotation for the J01002 from the plates indicated it was pSB1AC3. That would be bad--it needs to be just ampR. So, we grew up the two different J01002's from the freezer stocks (Box1>6v4) and (Box1>8v2). The second one for some reason is not in the slot indicated on the -80 stocks page. Someone will need to re-update the -80 stocks page. Dan spotted the two J01002 strains and J01064 from Box1>5v1 (oriTF/OnRFP). All three grew spots on amp but not on Cm or Kan. So, they are all 3 on pSB1A* plasmids, so it's all good. Incidently, J01064 was flaming pink in cell pellets. Much brighter than J01024 despite them having the same RFP cassette. I miniprepped J01024 and J01064.
JL_06/07/06
Goal: We want to determine the antibiotics resistence of the F and R knockout plasmids (TlambdaOlambda)
Results:
We grew TlambdaOlambdaRlambda to early log density. We then streaked the cultures with pipette tips on LB agar plates containing the following anti-biotics:
- 15ug/mL gentamicin (G)
- 50ug/mL ampicillin (A)
- 25ug/mL chloramphenical (C) ---errr, axe that datapoint, the plate disapeared.--JCAnderson 13:45, 8 June 2006 (EDT)
- 50ug/mL spectinamycin (Sp)
- 25ug/mL tetracycline (Tet)
- 20ug/mL trimethylprim (Trim)
TlambdaOlambda grerw on kan only
Rlambda grew on kan and tet only--JCAnderson 19:07, 7 June 2006 (EDT)