"
Team:Bordeaux/NotebookJuly10
From 2014.igem.org
(Difference between revisions)
(Created page with "{{:Team:Bordeaux/Tete}} <html> <h1>10<sup>th</sup> July </h1> <div class="type-exp"> Molecular biology</div> <br> <ul class="day-detail"> <li>Gene SLP synthesis<br><br> Step ...") |
|||
| Line 8: | Line 8: | ||
<ul class="day-detail"> | <ul class="day-detail"> | ||
| - | <li> | + | <li>SLP gene synthesis<br><br> |
Step 2:<br> | Step 2:<br> | ||
Latest revision as of 02:26, 18 October 2014
10th July
Molecular biology
- SLP gene synthesis
Step 2:
We made 35 cycles for the PCR instead of 30.
We made again the calcul of the annealing temperature with the estimation of the concentration in fragment after the first step.
We extended the time let for the hybridation.
PCR parameter:
92°C 1min
35 cycles : 92°C 10s, Gradient 64°C delta 6°C : (2AB) (3) 62°C 1min ; (2CD) (11) 66°C 1min, 72°C 10s
End at 4°C
PCR Parameter :
98°C 5 min
35 cycles : 98°C 10s, Gradient 61°C delta 18°C : (2AB) (11) 67°C 20s ; (2CD) (12) 68°C 20s (instead of 69°C), 72°C 1min
Final extension 72°C 10min
End at 4°C
We obtain this gel
Step 3: Vtot = 100µL and then 2x50µL
Estimation of the quantity with the gel to calculate the annealing temperature (Tm)
20µL phusion buffer 5X
0,5µL dNTP 10mM
1µL phusion polymérase
3,5µL DMSO
10µL 2AB8°°
10µL 2CD10°
55,5µL H2O
PCR parameter:
92°C 1min
35 cycles : 92°C 10s, 66°C 30s, 72°C 10s
End at 4°C
Gel 3% 45min 75V 10µL + 1,1µL
Production and purification
Please look our planning of production and purification
