Samantha Liang Notebook2
From 2007.igem.org
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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. <br> | 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. <br> | ||
Here's a picture of the gel: | Here's a picture of the gel: | ||
| - | [[Image:SIL072007gel.jpg]] | + | [[Image:SIL072007gel.jpg]] <br> |
| - | + | 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. | |
*Miniprep 408C, 408E, and 411B (by swabbing it) | *Miniprep 408C, 408E, and 411B (by swabbing it) | ||
*run PCR on 411B (ca998/G01001) and gel purify it | *run PCR on 411B (ca998/G01001) and gel purify it | ||
| - | *Sequence 408C, 408E | + | *Sequence 408C, 408E |
*Make -80s of 408C and 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. | *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. | ||
Revision as of 00:03, 21 July 2007
My Construction Files
My Sequencing Files
My Biobricks
My -80 Stocks
First Notebook (June - July 19, 2007)
For tomorrow:
- 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)
- Pool I716430 through I716435
- Analyze sequencing results
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.
- 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.
