Team:Paris/20 August 2009
From 2009.igem.org
(→Lab work) |
(→Lab work) |
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Digestion in XP | Digestion in XP | ||
- | A30->D40 | + | A30->D40 (Nter RFP) |
- | A31->D41 | + | A31->D41 (RFP) |
- | A33->D42 | + | A33->D42 (Cter RFP) |
with: | with: | ||
Line 69: | Line 69: | ||
-5µL Buffer 2*10 | -5µL Buffer 2*10 | ||
-20,5µL H20 | -20,5µL H20 | ||
+ | |||
+ | Then purification | ||
</div> | </div> | ||
Line 76: | Line 78: | ||
<div class="experience"> | <div class="experience"> | ||
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) | ||
+ | </div> | ||
{{Paris2009_Calendar_Link|19_August_2009|21_August_2009}} | {{Paris2009_Calendar_Link|19_August_2009|21_August_2009}} |
Revision as of 22:33, 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)