Lab Aug 24 2009

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)

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