Team:Caltech/Week 6

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Caltech iGEM 2011

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July 18

Colonies 1-6 from Gibson plates; 7 and 8 are reswabs of two colonies. From left to right: DNA ladder,(PNT001; colony 1; vector), P2v, P3v, P4v, P5v, P6v, P7v, P8v, (PNT001; colony 1; insert), P3i, P4i, P5i, P6i

from left to right: DNA ladder, (PNT001; reswab 7; insert), P8i, (16s; colony A; vector), 16Bv, 16Cv, (16s; A; insert), 16Bi, 16Ci

Colony PCR of colonies from positive Gibson plates from Friday, for vector and insert

Colony PCR of 16s plasmids, for vector and insert
Gel of short PCR parts to confirm they are being amplified
Pulsed field gel with a larger amount of soil sample DNA
Retransform pSB3K5 into Xl-10s

Results

Gels show mostly expected single bands

July 19

PCR parts for Gibson assembly of pNT001, pNT003 and Joe's positive GFP control

Results

pSB3K5 plate had 0 colonies.
PFGE of soil-extracted DNA shows a smear; will do gel extraction of ~40kbp sequences for fosmid kit.

lane 1 lambda mono-cut ladder, 2 blank, 3 40 kb control DNA, 4 LA River sample 9, 5 LA-River sample 10

Gibson PCR concentrations

Part	Concentration (ng/ul)
R0010 for pNT001	86.4
B0014 for pNT001	128.3
B0014 for pNT003	89.1
promoter for GFP Gibson positive control	51.9
GFP for positive control	130.4
terminator for positive control	82.5

@@ Line 17: / Line 17: @@
 ===Results===
 pSB3K5 plate had 0 colonies.<br/>
-PFGE of soil-extracted DNA shows a smear; will do gel extraction of ~40kbp sequences for fosmid kit.
+PFGE of soil-extracted DNA shows a smear; will do gel extraction of ~40kbp sequences for fosmid kit.<br/>
+[[File:pfge7192011.jpg|thumb|lane 1 lambda mono-cut ladder, 2 blank, 3 40 kb control DNA, 4 LA River sample 9, 5 LA-River sample 10]]
 Gibson PCR concentrations