Team:Paris/August 8

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Minipreps : Plasmid extraction

Extraction of pSB3K3 et E0240 in pSB1A2 plasmid from overnight bacteria culture using the QIAspin Miniprep Kit (QIAGEN) by QIACube.

Carried out 2 times (2 tubes)

name	Biobrick	plasmid
MP142	-	pSB3K3
MP143	E0240	pSB1A2

Transformation results

Name	Description	Antibio	Number of colonies	Number of red fluorescent colonies
Ligation
L128	J61002-pFlgA D136 (FV) - D132 (FI)	Amp	~ 400	2
L129	J61002-pFlgB D136 (FV) - D133 (FI)	Amp	39	5
L130	J61002-pFlhB D136 (FV) - D134 (FI)	Amp	~ 1000	4 (but 3 are on the edge of the petri dishe)
L131	J61002-pFlhDC D136 (FV) - D135 (FI)	Amp	39	38
Control
Control 1	D136	Amp	0	0
Positive control	pUC19	Amp	36	0

PCR Screening of Ligation Transformants

Use of 8 clones of Ligation transformants for screening PCR

Border="1"

Ligation

Name

n° clone

fluorescence

L128

pFlgA

OligoF_VF2 (O18)

1µl

10µM

OligoR_VR (O19)

1µl

10µM

water

23µl

Protocol of screening PCR

Mix

Name	Vol (µl)	Concentration
Quick Load	25µl	2X
OligoF_VF2 (O18)	1µl	10µM
OligoR_VR (O19)	1µl	10µM
water	23µl

50µl of Mix PCR by tube/clone
one toothpick of each clone's colony by tube
Program : Annealing 55°C - Time élongation 1'30" - Number cycle : 29

Conditions of electrophoresis

10µl of ladder 100 pb
10µl of screening PCR
migration ~30min at 100W on 1,5% gel

Results for L100

L100= D110 + D130 = RBS-tetR-ECFP-Ter

Preparation of the newly ammplified promoters

Electrophoresis of the PCR products made yesterday

Standart PCR to amplify pflgA, pflgB and pflhB(Gel1)

PCR with gradient to amplify pflhDC(Gel2)

Electrophoresis settings

Gel : 1.5 % agar
3µL template DNA
10µL QuickLoad DNA ladder 100 bp

Name	Promotor	Gel	Band	Expected size	Measured size
PCR_124	pFlgA	1	2	261 pb	250 pb
PCR_125	pFlgB	1	3	261 pb	250 pb
PCR_126	pFlhB	1	4	260 pb	250 pb
PCR_127	pFlhDC	2	2 to 13	446 pb	nothing

Results

We have no results for pflhDC, wo don't know yet where is the problem. We will try with other conditions! (yet undetermined)
Concerning pflhB, pflgA and pflgB, the protocol seems to be very operational: we always have great results !