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Tuesday July 5th, 2016

Tuesday, 7/5

PMembers Present: Kat, Celine, Karim, Marc, Bohdan

LAB:

Morning:

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CCD cells transformed with miniprepped RFP. Note overgrowth that is superior to CCC cells

CCC cells transformed with miniprepped RFP. Note overgrowth.

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CCD transformed with 50pg of RFP. 116 colonies

CCD transformed with 0.5pg of RFP. No colonies.

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CCD transformed with 10pg of RFP. 21 colonies.

CCC cells transformed with N5 ligated construct (new backbone - sample 5 PCR LacZ) . No colonies.

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CCC cells transformed with N2 ligated construct. This is the new PCR'd backbone with the two step PCR LacZ construct. No colonies

CCC cells transformed with O5 ligated construct. The O5 refers to the old PCR backbone with the sample 5 PCR version of the LacZ construct. 1 colony.

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CCC cells transformed with O2 ligated construct. The O2 refers to the old PCR backbone with the two step PCR version of the LacZ construct. 2 colonies.

Comparison of CCC and CCD transformed with RFP. Note increased growth on the CCD versus CCC plates.

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iGEM - july 5 - CCC CCD RFP plates.jpg

CCC (left) and CCD (right) cells transformed with RFP visualized under UV light. Note the increased growth on CCD versus CCC.

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(gradient) PCR amplified more pSB1C3 backbone from miniprepped RFP

Afternoon:

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PCR purification of PCR amplified backbone.

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Nanodrop of PCR purified constructs:

	A	B	C	D
1	Sample ID	260/280	260/230	concentration
2	74.4	1.62	0.76	62
3	72.4	1.92	2.26	23.3
4	70	1.53	0.69	21.2
5	68.1	1.5	0.57	18.6

Table1

PCRPur-BB-74.4.TIF

Nanodrop of 74.4C amplified backbone.

PCRPur-BB-72.4.TIF

Nanodrop of the 72.4C amplified backvone.

PCRPur-BB-70.TIF

Nanodrop of 70C amplified backbone

PCRPur-BB-68.1.TIF

Nanodrop of 68.1C backbone.

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Gel of PCR amplified backbone. Also included lanes with miniprepped E.glowli and L1 (first ligated construct)

iGEM - july 5 - PCRgrad-BB.jpg

GEL of gradient PCR of pSB1C3 backbone and miniprep products:Lane 1: 74.4C Lane 2: 72.4C Lane 3: 70C Lane 4: 68.1C Lane 5: 3K Ladder. Lane 6: Miniprepped L1 construct. Lane 7: Miniprepped E.glowli construct.

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5ml overnight cultures of RFP, O5, O2 for miniprep tomorrow.

TO DO:

For the next day:

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Miniprep of RFP, O5, O2

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Gel of miniprepped products to ultimately determine which ligated variant is superior. This is to determine which method of PCR is superior -> two step PCR or the generic variant. Once this is determined, we can proceed with PCR amplifying the rest of our construct.

Team:Toronto/Notebook-w08-tue