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Team:Paris/20 August 2009
From 2009.igem.org
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| - | Ligation | + | '''Ligation''' |
| - | DO: | + | '''DO:''' |
-PSB1A3: 0,61 µg/ml | -PSB1A3: 0,61 µg/ml | ||
| Line 88: | Line 88: | ||
-D42:0,40 µg/ml | -D42:0,40 µg/ml | ||
| - | D40 with PSB1A3 in 3X and 10x | + | '''D40 with PSB1A3 in 3X and 10x''' |
For 3x(3,7µl insert+1µL vector+1µL buffer 10*+1µl ligase+3,3µL H2O) | For 3x(3,7µl insert+1µL vector+1µL buffer 10*+1µl ligase+3,3µL H2O) | ||
| Line 94: | Line 94: | ||
For 10x(12µl insert+1µL vector+2µL buffer 10*+1µl ligase+4µL H2O) | For 10x(12µl insert+1µL vector+2µL buffer 10*+1µl ligase+4µL H2O) | ||
| - | D41 with PSB1A3 in 3x and 10x | + | '''D41 with PSB1A3 in 3x and 10x''' |
For 3x(2,5µl insert+1µL vector+1µL buffer 10*+1µl ligase+4,5µL H2O) | For 3x(2,5µl insert+1µL vector+1µL buffer 10*+1µl ligase+4,5µL H2O) | ||
| Line 100: | Line 100: | ||
For 10x(7,5µl insert+1µL vector+1,5µL buffer 10*+1µl ligase+4µL H2O) | For 10x(7,5µl insert+1µL vector+1,5µL buffer 10*+1µl ligase+4µL H2O) | ||
| - | D42 with PSB1A3 in 3x and 10x | + | '''D42 with PSB1A3 in 3x and 10x''' |
For 3x(3,7µl insert+1µL vector+1µL buffer 10*+1µl ligase+3,3µL H2O) | For 3x(3,7µl insert+1µL vector+1µL buffer 10*+1µl ligase+3,3µL H2O) | ||
| Line 106: | Line 106: | ||
For 10x(12µl insert+1µL vector+2µL buffer 10*+1µl ligase+4µL H2O) | For 10x(12µl insert+1µL vector+2µL buffer 10*+1µl ligase+4µL H2O) | ||
| - | For negativ control: | + | '''For negativ control:''' |
0µl insert+1µL vector+2µL buffer 10*+1µl ligase+7µL H2O | 0µl insert+1µL vector+2µL buffer 10*+1µl ligase+7µL H2O | ||
Latest revision as of 22:36, 16 October 2009
NoteBook
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Lab work
PCR w/ Skywalker
Pcr of:
A35 - FecI from pSV26 Worked
A35 bis- FecI from K12 genomic DNA source Failed (Skywalker is only a padawan)
Purification of Pcr product
directly after PCR:
IMAGE
After purification :
IMAGE (wait until tomorrow)
Digestion
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
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
