Berk2006-Samantha's Notebook

9-06-06

• culture of pSB3C6-J23072 clone #1 was slightly pink, so I proceeded only with clone #2 which was white and should be correct.
• Miniprepped, made a -80, and sequenced pSB3C6-J23072-2. Also threw out pSB3C6-J23071-1 from -80 stocks, because clone 2 was the right one.
• transformed J23072-2 and J23071-2 into J01129 (Chris will put plates in fridge tomorrow)
• all cultures of pSB3C6-I13521 appear white, even after pelleting, they still look white, so something must have gone wrong.

9-06-06

• grew up two white colonies of pSB3C6-J23072
• sequencing for J23071 clone#2 looks good and is in the -80
• pSB3C6-I13521 plate has all white colonies it seems unfortunately. Growing up four clones just in case they turn pink in culture.

For Friday:

• miniprep clones of pSB3C6-J23072, make -80s, and send them in for sequencing
• transform J23072 and J23071 into J01129
• Pick colonies Sunday and Assay on Monday

8-25-06

Riboregulators:

• cultures of new locks J23071 and j23072 look a little red. Miniprepped 2 white cultures of pSB3C6-J23071, made -80 stocks of them, and sent them for sequencing with ca1000. Pick colonies of J23072 again when we get back from the break, plates are in the fridge.
• ligated and transformed pSB3C6-I13521. (John will put in fridge tomorrow)

Conjugation:

• purified the PCR product of pSB1A2-J23038 and digested with BsaI/SpeI/dPN1, then purified again.
• digested pSB1AK3-J23026 (traG+TT) and pSB1AK3-J23028 (trbCf+TT) with BsaI/XbaI, ran gel and gel purified.
• ligated and transformed to get products pSB1AK3-J23074 ([pCon][RBS][traGr][TT]) and pSB1AK3-J23075 ([pCon][RBS][trbCf][TT]). (John will put in fridge tomorrow)

8-24-06

Riboregulators:

• grew up 4 white colonies each of new locks J23071 and J23072.
• Started to make pSB3C6-I13521 to replace pSB3C6-J01022, which just removes the upstream Ptet to do future measurements relative to a more-similar standard. Made a construction file and cleaned up digestions of I13521 and pAC997 (found in "Good" Plasmids box). Ligate and Transform tomorrow.
• sequenced pJ23006-J23070 #1 and pJ23006-J23062 #1 (the tRNA-based keys)with ca998.

Conjugation:
Finishing up the traG and trbC complements

• PCRed pSB1A2-J23038 with G00101 and ca641R to get the short pCon+RBS fragment. Tomorrow, digest this fragment with BsaI/SpeI/dPN1. Also, digest pSB1AK3-J23026 (traG+TT) and pSB1AK3-J23028 (trbCf+TT) with BsaI/XbaI. Purify, ligate and transform to make new pSB1AK3 complements. Make construction files. BsaI is not in the APE enzyme list it is GGTCTC.
  

--Samanthaliang 17:04, 24 August 2006 (EDT)