| 
  • If you are citizen of an European Union member nation, you may not use this service unless you are at least 16 years old.

  • You already know Dokkio is an AI-powered assistant to organize & manage your digital files & messages. Very soon, Dokkio will support Outlook as well as One Drive. Check it out today!

View
 

Lab aug 24 2009

Page history last edited by Layne Woodfin 14 years, 7 months ago

Derek is coming in at 2 to make competent cells, layne can also be in at 2. I'm here now...

 

Today we could run out our digests from the 20th on a gel to see if they are alright, and if so try again with the ligation and transformation steps

 

Also from what I can tell right now some of our parts from the first assembly are questionable.

 

we need to run out  K235000-200ul plate digest to see if there is a band, as the 100ul plate didn't produce one.

 

We probably should have run a denser gel for the digested parts, as anyrthing less than 500bp probably ran off the gel, eh?

 

Also when we did the calculations for the undigested plasmid we would have been comparing linear DNA from the ladder to circularized DNA from the plasmid, making our sizes not quite right? Yeah, just checked with Melissa and we can't use that data.

 

Having gone through all that, I think the best call for today is to hit our parts with EcoRI and then run them out on a .7% gel for slightly better resolution. I'm not going to recomend doing more assemblies until we have some useful data on the results of this one.

 

 

 

Doing:

 

Making a .7% gel (0.42g agrose)

 

Digesting k23000-002 with EcoRI, in 37 bath at 12:55, 80 bath at 1:10

 

Gel-starter at 2:20pm

 

Ladder K235000(100ul) k235000(200ul) k235001(100ul) k235001(200ul) k235002(100ul)
Bands No band No band Band Band Band
Size *3171 *3101 2425 2508

2965

2652

aug 24 gel 1.tif 

 

- Layne

 

* On closer examination of the .tif file, I could just barely make out two faint bands in each of these lanes that were almost identically position on the gel.

Given the faintness I don't think we should pay much attention to the discrepency between sizes in lanes 2 and 3.  It was probably due to my error.

 

You can look at my calculations here.

 

 

Competent cells:

 

we are having SUCH a hard time calibrating the incubation duration and conditions to get to an OD of .3

 

After 16 hours of room temperature (cool room) and minimal agitation the OD was only .04

Previously, of course, it went up over OD .6 in an overnight with full agitation.

I've stuck the culture in the fridge and will bring it back out tomorrow while we work on other things to do it's last few hours of agitated room temperature growing up.

 

 

Gel problems:

 

Thinking about this some more, I  think we need more data points. Fran originally suggested picking a whole bunch of colonies, so I've gone back to our successful composite parts and picked some more for overnight culture tonight. I  picked 2 from K235000, 1 from K235001, and 2 from K235002. Hopefully we can miniprep and gel these tomorrow to see if the products look the same.

 

Things to do

 

- miniprep and gel additional composite part picks

- competent cells

- Lab space discussion with profs

- K235003-K235005 didn't grow but the extended 37oc step is an obvious reason so we can just do them again

- We need more Cm plates made

- k145303 and k235001 might make a good test assembly but do we need kan plates to do it?

 

Gels that have not been run:

- K145303 Quad (miniprepped)

- P1010 Cm Plasmid digest (not enough room)

- K098988 and K081005 (miniprepped)

- R0010 (pLac), J23066(ribokey + terminator), C0051 (λcI repressor), I13521 (Ptet RFP generator) (was miniprepped but not run)

Comments (1)

Chris Tuttle said

at 4:13 pm on Aug 24, 2009

"I'm not going to recomend doing more assemblies until we have some useful data on the results of this one."

Actually, I'm not sure agree with this. It seems like with one more set of assemblies we can have something testable in a way other than the gel data. Given that P1010 Kan has never shown a band, even from the orginal rehydration and we know it transformed our cells because it introduced Kan resistance, I'm suspicious of our gel protocol still. I know we need to debug that, and in the long term that's first priority. At the same time I'd really like to see a combined product do something that we can visualize.

None of our composite parts do this on their own. We do have decent bands for K235001, though. (That's the constitutive promoter and the ribokey with stop) Among our rehydrations is K145303 (the promoter and ribolocked GFP part). If we were to do use our existing downstream digest of K235001 compbined with an upstream digest of K145303 then we would have something that we could confirm on the flow cytometer while we work on the gel problems.

I think it's worth another digest and ligation to see if we can get that to work. Hopefully in parallel with the investigation of the gel problems.

You don't have permission to comment on this page.