Samantha Liang Notebook2

From 2007.igem.org

Revision as of 23:16, 24 July 2007

My Construction Files
My Sequencing Files
My Biobricks
My -80 Stocks

First Notebook (June - July 19, 2007)

For tomorrow:

• Make large scale competent cells of Righty and Lefty
• Pick colonies if I716436 (1106A.Cre) and I716437 (1109.Cre)
• Pool I716430 through I716435 so you have minipreps of them

Samanthaliang 19:09, 24 July 2007 (EDT)

• Subcloned 1106A.Cre and 1090.Cre (rbs.Cre)
• PCR with ca56(in pBad promoter)/G01001 on minipreps of 416, 417, 418, 401, 411, and 412 to examine the size of the cassette - these are the ceaB variants and also a barnase and 2 bglII variants that showed some activity in the initial round of tecan screening
• Colony PCR of inactive (or rather less active) 408s - 408A, 408B, 408D, 408F
• The wells that I grew yesterday with the RFP reporter mechanism did not turn red at all so I'm going to examine that some more.
• Colony PCR of of 434 (GTG TT verision of RFP reporter) - 4 clones to look at cassette
• Chris saw that rbs.atg.bamH and rbs.atg.BglII wasn't lethal in Lefty, Righty, and DH10B - but will be checking if it is lethal once he adds a promoter

Samanthaliang 21:28, 23 July 2007 (EDT)

• Pick colonies from I716430 through I716435 in a 96 well block (swirl each colony in a well with arabinose and a well without) then check if the cultures turn red (might even be able to run a quick tecan on it to see the RFP levels)
• Analyzed sequencing results for 408C and 408E - unfortunately, these two guys are the same thing - [pbad][rbs][barnase - no ATG], which is not what I want because it is missing both Cre and the cassette. This must have occurred by some crazy recombination or something. I need to do some PCR mapping on some other clones to troubleshoot. I think hits are just falling out like crazy. Maybe it'd also be helpful to make libraries on low copy plasmids? Might be able to pick up more hits.
• Long google chat with Chris about next steps

Samanthaliang 15:17, 20 July 2007 (EDT)

Things to read about:

• Does barnase have DNase activity? Haven't really found anything saying that it has DNase activity yet, but it is a ribonuclease that destroys all the RNA and targets the ribosome, which in turn, stops DNA synthesis such that those bacteria should no longer be able to replicate and grow.
• Is there a way to do dapi (pronounced dap-E) measurements for e.coli? dapI binds to DNA, wonder if can use a cytometer or something? Do a dapi stain? YES! Can do a dapi stain in e.coli and then do measurements with a flow cytometer.
• Make sure that we're not just making a toxic protein or that Cre itself is toxic or something like that.

So I'm having a lot of trouble isolating the plasmid of 411B. The plate that I got from streaking a 96 well culture has really sickly single colonies that are super tiny. I can't get any of the colonies that I pick to grow in liquid media even when I add glucose. It also didn't grow after being streaked onto another plate. It seems like that in order to try to isolate a plasmid and sequence it, I'm going to have to take a swab of the plate in a dense area and miniprep that (put it in p1 buffer directly) then I'm going to have to sequence off of a PCR product.
Here's a picture of the gel:
Unfortunately, there were no bands on it, meaning that either that part isn't what I think it is, or else, all the cells are already dead and the primers weren't able to pick up anything big enough to see on the gel from the plasmid. I can also see kind of a smear, which could mean that I'm picking up junk, but more likely, it's just some discoloration from the dye or something.

• Miniprep 408C, 408E, and 411B (by swabbing it)
• run PCR on 411B (ca998/G01001) and gel purify it
• Sequence 408C, 408E
• Make -80s of 408C and 408E
• Look at spotted plates - the spots actually look like they're less dense for the ones that showed a plateauing growth rate. I don't think it's obvious enough to be convincing of anything though. I guess if any cell is alive though, it would grow in the spot.