Team:Caltech/Week 8

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Caltech iGEM 2011



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July 31

Started a LB+amp overnight culture of the B0034 glycerol stock from the 2010 Caltech iGEM Team

Results

Mass spectrometry did not run correctly; samples did not ionize; must rerun

August 1

PCR pSB3K3 and pNT003 insert with the new primers.
Gel extract the pNT002 insert from the July 29 PCR
Minimal media transfers
Transformation and plating of pUC19 into competent cells for cell dry weight experiment
Overnight culture of B0034 glycerol stock
Overnight ligation of pNT002 insert and PSB3K3 backbone
Restriction digest of parts for pNT002 standard assembly and overnight ligation

Results

No colonies visible on the pUC19-transformed cells

The enrichment culture plates show no visible growth yet.

Gel Extractions of July 29 PCR of pNT002 and pNT003 inserts

Part Concentration (ng/ul)
pNT002 insert 65.9
pNt003 insert 12.5

August 2

Gibson assemble pNT002 using the gel extracted insert and linearized pSB3K3
Gibson assemble pNT003 using the gel extracted insert and linearized pSB4A5
Transform Gibson reactions into our XL-10 cells and Emzo's top ten cells
Replating of more pUC19-transformed competent cells and non-transformed competent cells
Standard Assembly Digest- K12300, B1004, R0010, B0034, along with the destination plasmids.
Run gel of overnight pNT002 ligations
Transformed pNT002 insert+ pSB3K3 ligation

Results

pNT002 ligation showed band on gel

Part Concentration (ng/ul)
pSB3k3-1 23.8
pSB3K3-2 29.9
pETDEST53 90.9

August 3

Colony PCR of Gibson transforms

Results

Plate number of colonies
pNT002 + top ten 55
pNT002 - top ten 43
pNT002 +XL10 gold 180
pNT002 - XL10 gold 400
pNT003 + top ten 14
pNT003 - top ten 1
pNT003 +XL10 gold 0
pNT003 - XL10 gold 7

August 4

Ran gel of colony pcr from yesterday

Results

Some of the colony PCR have insert amplifications of the correct length. Will send some off for sequencing tomorrow.


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