Berk2006-ConjugationTeam

JL_06/22/06
Today we mapped pSB1A2-J01024 using EcoRI and SpeI and pSB3C6-J01093 BamHI and SpeI. We had 100% recovery of 4 clones of each, and did not get any colonies of just the backbone alone.
We also digested, gel purified, ligased, and transformed JO1001 and B0015 to get the product J23003 (TraJf-tt).

SIL_06/19/06
Today we subcloned pSB1A2-J01024 into the pAC997 backbone to create the final product pSB3C6-J01024. We did this by digesting pSB1A2-J01024, gel purifying it, ligating it, and then heat shocking it. We also subcloned pSB1A2-J01093 into the pAC997 bacbone to create pSB3C6-J01093, starting from a PCR product of J01093 because in a previous experiment J01093's results came out unclean. This was also done through digestion, gel purification, ligation and heat shock.
We also restreaked Rlambda from the -80 freezer stocks onto a Kan plate.
Lastly, we made sure that J01001 was in the freezer for future use.
--Samanthaliang 19:17, 19 June 2006 (EDT)

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:

1. 15ug/mL gentamicin (G)
2. 50ug/mL ampicillin (A)
3. 25ug/mL chloramphenical (C) ---errr, axe that datapoint, the plate disapeared.--JCAnderson 13:45, 8 June 2006 (EDT)
4. 50ug/mL spectinamycin (Sp)
5. 25ug/mL tetracycline (Tet)
6. 20ug/mL trimethylprim (Trim)
   

TlambdaOlambda grerw on kan only
Rlambda grew on kan and tet only--JCAnderson 19:07, 7 June 2006 (EDT)