Team:Paris/17 August 2009

NoteBook
August 17th

 

Lab work
Competent Bacteria


 * Competent bacteria
 * 1/50 ON culture of New DH5&alpha;
 * OD600=0,6
 * Transformation with puc19 (10pg/µl)
 * 250µl of competent DH5&alpha;
 * 1µl puc19
 * Leave 20 min in ice
 * 45s at 42°C
 * Leave 2 min in ice
 * Add 1ml of hot LB (42°C)
 * 1h under agitation
 * Put on plate (LB agar + Amp)

Vector Dephosphorylation

pSB1A3 digested by EcoRI/PstI, XbaI/PstI and EcoRI/SpeI have to be dephosphorylated before the ligation process.

Mix : Vector : 30uL

Antarctic Phosphatase : 2uL

Buffer : 3,5 uL

Protocol

Put the solution at 37°C for 30 min

Add 2 uL of enzyme

37°C for 30 min

65°C for 20 min for heat inactivation

Ligation

D13 (X/P): 0,94 ug/mL

D14 (X/P): 1,2 ug/ml

pSB1A3 (X/P): 2,1 ug/ml

Ligation (2hrs at RT) of pSB1A3 w/ D13 (ompA signal) or D14 (TolRII)

Mix : total volume = 10 uL

vector (pSB1A3) : 2 uL

insert (A11 or A13) : 4uL

10X T4DNA Ligase buffer : 1 uL

T4 DNA Ligase : 0,5 uL

H2O: 2,5 uL

Negatif control : same mix as the previous one but without the insert. In these conditions the amount of water was increased until 6,5 uL.

Transformation

Transformation of (using the "heat choc" protocol) :

- BBa_B0034 (AmpR)

- BBa_E0030(KanR)

- pSB3C5 (CmR)

- pINTE3 (Col E3, AmpR)

- pTA (pTet-Col A, AmpR)

- pINTg3p (g3p Term, AmpR)

- pINK2(TolRII, AmpR)

- Ligation 1 ( set up on Friday 14th of august w/ D11 (RBS-Tet digested by Xba/Pst) and vecteur D12 (pSB2K3 w/ pLac digested by Spe/Pst) (KanR)

- previous ligation (w/ D13 and D14) (AmpR)

Transformation Check/PCR on colonies Transformation check:

The x10mix ligation after centrifugation and put at 16°C overnight had work.

using less incubation time at RT seems to work better.

using less insert seems to work better

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 3