Team:Paris/18 August 2009

NoteBook
August 18th

 

Lab work
PCR on colony w/ Soufifi

The ligation pLAC/RBS-TetR worked --> no clone on the negatif control and some clone on the "good" plates. PCR on colony is required to check the validity of our clones.

Mix : total volume = 25uL

Super Mix 2X: 12,5 uL

VF2 (10uM): 0,5 uL

VR (10 uM): 0,5 uL

H20 : 6,5 uL

diluted bacteria : 5 uL

Tm = 55°C

Elongation time = 1 min

size expected : 

- pLac/RBS-TetR : +/- 700 bp

- VF2 and VR plasmid amplification : +/- 150 bp

- final size : +/- 850 bp



Digestion The ligation that I have set up yesterday (pSB1A3 w/ D13 or D14 ) didn't work so I decided to redigest PCR products (A11 (RBS-ompA signal = D13) and A13 (TolRII = D14)).

Mix : total volume = 30 uL

For A11 (RBS-OmpA signal):

DNA = 15 uL

10X buffer: 3 uL

SpeI: 1 uL

PstI: 1 uL

BSA: 0,5 uL

H2O: 9,5 uL

For A13:

DNA = 10 uL

10X buffer: 3 uL

XbaI: 1 uL

PstI: 1 uL

BSA: 0,5 uL

H2O: 14,5 uL

Other digestions were done : 

- The double terminator (BBa_0034) by EcoRI/XbaI and SpeI/PstI (same mix as the one for A13)

- The pTet (BBa_....) by SpeI/PstI and EcoRI/PstI

Gel purification w/ Bobode

Promega kit, 3 uL loadded



Gel purification

PCR on colonies:

using for 1 tube:

Master Mix 2X : 12,5µL

Oligo VF2 (10µM): 0,5µL

Oligo VR (10µM): 0,5µL

bacteria in H20 (see PCR on colonies protocol): 5µL

H20 : 6,5µL

PCR on colonies Check OK for colonie 7, 8, 9 and 10

Control+ (with PSB1A3)

Colonie 7, 8 and 10 OK.

Stock Glycerol

stock glycerol for colonie 3 ClyA in PSB1A3 using: -1/3 glycerol (333µL) + 2/3 bacteria (667µL)

Miniprep for colonie 3, 7, 8 and 10

Miniprep for colonie 3, 7, 8 and 10