Lab Aug 21 2009

Rats, no growth last night! Well, there are some tiny pinprick colonies tht may turn into something, but it's not the lush plate I was hoping for.

Also, someone took my temperature sensitive GFP that was suppoosed to be in the 42 degree overnight and put it in the fridge! Double rats!

I'm spinning down some Part "C" to see if the ECFP is visualizable in the concentrated cells.

Can't really see anything in the spun down cells. I went and looked at some of them under the microscope, and using the fluorescent microscope, but the fluorescent scope doesn't have filters that are quite right so that was inconclusive. (The filters will excite down to 450 nm but we need 440.)

Layne is working on minipreps from last night.

And he's done!

plasmids for 100 and 200ul versions of K235000, K235001 and K235002 (6 total).

Gel 1 (whole undigested plasmids) started at 12:05 1 	2 	3 	4 	5 	6 	7 	8 Ladder

K235000

100ul plate

K235000

200ul plate

K235001

100ul plate

K235001

200ul plate

K235002

100ul plate

K235002

200ul plate

K235002

200ul plate

8 ul H20 Approximate size (bp) 	2608 	2608 	2443 	2443 	3024 	3024 	3024
 * Observed size 	2517 	2642 	1921 	1921 	5043 	2330 	2330

Aug 21 gel 1.tif


 * Layne - You can look at my calculations here.

Cropped and highlighted gel: Aug 21 gel 1 modified.tif Aug21 gel 1 modified version.jpg

I highlighted the bands in this one after the fact (not very accurately). Lanes 7 and 8 are actually significantly lower than 2 and 3.

(upstream part + downstream part + backbone = total size)

K235000 (200 + 336 + 2072 = 2608) seems to be the right size. (lane 3 at least, lane 2 could be missing something.....)

K235001 (35 + 336 + 2072 = 2443) is about 500 bp off, it could be missing the downstream part J23066 (ribokey + terminator).

K235002 lane 6 (35 + 917 + 2072 = 3024) is about 2000 bp off.......

K235002 lane 7 (35 + 917 + 2072 = 3024) is about 700 bp off, it could be missing the downstream part E0422 (RBS + ECFP + stop).

Performed restriction digests on K235000(100ul), K235001(100ul) and K235002(100 and 200ul).

All parts were treated as downstream parts (XbaI and PstI).

Gel 2 (digested parts from above plasmids + others) started at 2:52 1 	2 	3 	4 	5 	6 	7 	8 Ladder

K235000

100ul plate

K235001

100ul plate

K235002

100ul plate

K235002

200ul plate

P1010 Amp

miniprep

P1010

Kan

miniprep

P1010

Kan rehydration Approximate size (bp) 	536 	371 	953 	953

?

2-3kb ? 	?
 * Observed size 	No band 	2016

2316

1944	***1987 	2384 	No band 	No band

aug 21 gel2.tif


 * Layne - you can look at my calculations here.

Cropped and highlighted gel: Aug 21 gel 2 modified.tif Aug 21 gel 2 modified version.jpg.


 * I seem to have lost some reoslution in this image. At a second glance of the unmodified image, theres definately a band here.

We're thinking that the DNA ladder from Ivitrogen (this page) corresponds to our ladder.

Cryopreserved:

K235000

K235001

K235002 (Both versions, 100ul and 200ul because the band patterns appeared different...)

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