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Lab Aug 17 2009
From 2009.igem.org
Pre-lab, Kyliah
It occurs to me that the whole point of the ccdB genes is to kill the DH5α cells we transform things into after assembling pieces. We don't actually need a competent line of DB3.1 cells. We might be better served by ordering our failed P1010 parts as stabs, in addition to I0500 pAra, R0051 pLac, and whichever others have failed so far.
Chris
Sadly, the point is that we need CCDB containing biobrich compatible plasmid in large amounts for out assembly. Only way aside from buying it is to miniprep from cells containing the plasmid. I was thinking maybe today we could do an RFC10 assembly of the constituitive promoter and the ECFP tripart, in addition to transforming the CCDB resistant cells derek made competent on thursday (top10 protocol). they are in the freezer.
Natasha
We have plates with Kan p1010 and Cm P1010. Can we try purifying from the Cm plates, or is there a reason we haven't tried it yet? Also, should we try again from the Kan plate?
Chris
p.S. I'll be in around 10:45 11 today, and want to stay all day.
to Natasha. Nothing wrong with trying again from kan and trying the Cm plates, but it seems pretty likely, given that these colonies took far longer than usual to form and were tiny, that none of the colonies actually contain functioning plasmid. again, we can try for sure, but it's also pretty important that we do the transformation today.
Derek:
I think Ky's point is a valid one, actually. At least if my understanding of stabs is correct. Is a stab a stab of the growing E. coli expressing the part? If so then they would come on properly transformed cells.
I think the main thing today is the transformation.
I'm over in ECS right now. I'm working on a project and stupidly forgot my keys, so it's a bit of a pain to come back and forth, but I can chat by phone and come over at some point a bit later when someone else is here to let me back in. My phone number here is 250-472-5749
Natasha
Should we think about switching to the standard assembly to try to get past this roadblock?
For today, what transformation are we talking about?
11:00 am
Broth cultures started from P1010 Kan and Chl plates.
12:55 pm
Transformation Protocol Two transformations started for each frozen P1010 Kan and Chl rehydrated part.
1.30 pm
37oC incubation started.
4.00 pm
Plated 100 and 200 uL of each P1010 Kan and Cm
5:00pm - Kyliah
So, when should we order stabs (cultures of pre-transformed cells) and which ones? They should be delivered within two days of sending the request e-mail, and possibly even overnight. I think we should order I0500, P0051, P1010 Kan, and P1010 Cm. The P1010 Amp transformation should be miniprep'd and checked for true transformation.
* part BBa_I0500 - Inducible pBad/araC promoter - plasmid pSB2K3, kanamycin resistance - 2009 Kit Plate 1, well 14N or Spring 2008 Distribution, source plate 1013, well 5C * part BBa_R0051 - promoter (lambda cI regulated) - plasmid pSB1A2, ampicillin resistance - 2009 Kit Plate 1, well 6K or Spring 2009 Source Plates, source plate 2001, well 3F * part BBa_P1010 - ccdB cell death gene - plasmid pSB1K3, kanamycin resistance - 2009 Kit Plate 1, well 7I or Spring 2009 Source Plates, source plate 2000, well 4E * part BBa_P1010 - ccdB cell death gene - plasmid pSB1C3, chloramphenicol resistance - 2009 Kit Plate 1, well 5E or Spring 2009 Source Plates, source plate 2000, well 3C
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