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Lab Sept 02 2009
From 2009.igem.org
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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