Lab Sept 02 2009

Plans:

* Talk to Dr. Nano and Dr. Upton about where we'll have lab space during September * Talk to Allison about sequencing constructed parts * If any constructed parts have plasmid frozen but no cells frozen, retransform and culture * If any constructed parts have neither plasmid nor cells frozen, reconstruct and retransform cells

"EXTANT" PARTS Part # 	Upstream part 	Downstream part 	Destination plasmid 	Frozen plasmid? Frozen cells? Goal today K235000 	R0010(Amp) lactose promoter 	J23066(Amp) ribokey+terminator 	P1010-Cm 	yes 	yes 	none K235001 	J23102(Amp) constitutive promoter 	J23066(Amp) ribokey+terminator 	P1010-Cm 	yes 	yes 	none K235002 	J23102(Amp) constitutive promoter 	E0422(Amp) RBS+ECFP+stop 	P1010-Cm 	yes 	yes 	none K235003 	C0051(Amp) λcI CDS 	B0015(Amp/Kan) transcription stop 	P1010-Cm 	ligation product only 	no 	retransform? K235004 	R0010(Amp) pLac 	B0034(Amp) standard RBS 	P1010-Cm 	ligation product only 	no 	ignore for now K235005 	J06504(Amp) mCherry CDS 	B0015(Amp/Kan) transcription stop 	P1010-Cm 	ligation product only 	no 	retransform? K235006 	J23102 	(none) 	P1010-Kan 	ligation product only 	no 	Wait until Kan plasmid received K235007 	K145303 (Amp) 	(none) 	P1010-Kan 	ligation product only 	 no 	Wait until Kan plasmid received K235008 	K145303 (Amp) 	K235001 (Cm) 	P1010-Cm 	ligation product only 	 no 	forget we ever made this part K235009 	J23102(Amp) constitutive promoter 	J23032 (Amp) ribolock 	P1010-Cm 	ligation product only (parts failed) 	 no 	reconstruct from scratch K235010 	K145303(Amp) 	N/A 	P1010-Cm 	ligation product only 	 no K235011 	K235009(Cm) 	K235010(Cm) 	P1010-Kan 	none 	 no 	Wait until Kan plasmid received K235012 	K235011(Kan) 	K235000(Cm) 	P1010-Amp 	none 	no

Wait on K235011

decision for today:

- make new digest of P1010-Cm if we're running low (we were fine for today, there's about one or two rounds left)

- religate K235003, K235005, K235010 (renamed from former K235015)

- rehydrate J04450-Kan (plate 1, well 7A and plate 1, well 7G) - this is a constitutive (?) RFP generator on a Kan plasmid, we can use it instead of P1010-Kan for 3A assembly

- transform DH5α and plate on Kan (J04450) or Cm (K235***)

when we were transforming hte parts, the heat shock water bath was only at 40o instead of 42o

The stabs we ordered came in! We streak-plated all five of them. If P1010-Kan doesn't grow, no worries 'cause it's untested. If I0500 doesn't grow, then we can try replating it on a Kan plate that's had IPTG added in order to beef up the copy number. If I0500 DOES grow, make SURE that the broth culture has IPTG added to spike the numbers of plasmid.

Plates to check tomorrow

- K235003, K235005, K235010

- J04450-Kan 7A and 7G

- P1010 Kan, Amp and Cm

- I0500, R0051

We (Natasha and Kyliah) talked to Dr. Upton (we get use of bench space, use of bench equipment, 4o fridge, -20o freezer) and to Dr. Nano (we get use of -80o freezer). We should get ready to move on Thursday (we need boxes for our epis) and move late Thursday or early Friday.

- Talk to Erica about the flow cytometer for visualising GFP

- Get everyone applied to the wiki and then email Dr. Ishiguro - Derek and Kyliah are already done.

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