Team:Paris/20 August 2009

Pcr of:

A35 - FecI from pSV26 Worked

A35 bis- FecI from K12 genomic DNA source Failed (Skywalker is only a padawan)

directly after PCR:

IMAGE

After purification :

IMAGE (wait until tomorrow)

Digestion in XP

A30->D40 (Nter RFP)

A31->D41 (RFP)

A33->D42 (Cter RFP)

with: -20µL DNA -2µL Xba -2µL PST -0,5µL BSA*100 -5µL Buffer 2*10 -20,5µL H20

Then purification

Ligation DO:

-PSB1A3: 0,61 µg/ml

-D40:0,50 µg/ml

-D41:0,80 µg/ml

-D42:0,40 µg/ml

D40 with PSB1A3 in 3X and 10x

For 3x(3,7µl insert+1µL vector+1µL buffer 10*+1µl ligase+3,3µL H2O)

For 10x(12µl insert+1µL vector+2µL buffer 10*+1µl ligase+4µL H2O)

D41 with PSB1A3 in 3x and 10x

For 3x(2,5µl insert+1µL vector+1µL buffer 10*+1µl ligase+4,5µL H2O)

For 10x(7,5µl insert+1µL vector+1,5µL buffer 10*+1µl ligase+4µL H2O)

D42 with PSB1A3 in 3x and 10x

For 3x(3,7µl insert+1µL vector+1µL buffer 10*+1µl ligase+3,3µL H2O)

For 10x(12µl insert+1µL vector+2µL buffer 10*+1µl ligase+4µL H2O)

For negativ control:

0µl insert+1µL vector+2µL buffer 10*+1µl ligase+7µL H2O