"
Team:UNAM Genomics Mexico/Notebook/Protocols
From 2012.igem.org
(Difference between revisions)
| Line 14: | Line 14: | ||
<Center><h1>E. coli Protocols</h1></center> | <Center><h1>E. coli Protocols</h1></center> | ||
<br /> | <br /> | ||
| - | <h2>PCR | + | |
| + | <h2>PCR Protocol (50µl)</h2><br /> | ||
Buffer Pfu 5 µl<br /> | Buffer Pfu 5 µl<br /> | ||
Upper primers 2.5 µl<br /> | Upper primers 2.5 µl<br /> | ||
| Line 30: | Line 31: | ||
goto 2:4 30 times<br /> | goto 2:4 30 times<br /> | ||
72ºC 5 min<br /><br /> | 72ºC 5 min<br /><br /> | ||
| + | |||
| + | <h2>Hot start PCR protocol</h2><br /> | ||
| + | Preheated 105ºC<br /> | ||
| + | Heated Lid ON<br /> | ||
| + | Pause OFF<br /> | ||
| + | In denat 94ºC 5 min. <br /> | ||
| + | Hot start OFF<br /><br /> | ||
| + | 30 cycles<br /> | ||
| + | 94 ºC 30 secs. <br /> | ||
| + | 55ºC 30 secs. <br /> | ||
| + | 70ºC 45 secs. <br /> | ||
| + | 72ºC 5 min<br /> | ||
| + | hold 10ºC<br /> | ||
| + | |||
Revision as of 07:39, 14 September 2012
Protocols
E. Coli Protocols |
|
Bacillus Protocols |
E. coli Protocols
PCR Protocol (50µl)
Buffer Pfu 5 µl
Upper primers 2.5 µl
Lower primers 2.5 µl
MgCL2 1 µl
DNA (depends on concentration, usually 0.5µl)
Enzime Taq Polymerase 1 µl
DNTP’s 8 µl
Water 29.5 µl
Cycles :)
95ºC 4 min
95ºC 1 min
55ºC :30 secs
70ºC 1 min
goto 2:4 30 times
72ºC 5 min
Hot start PCR protocol
Preheated 105ºC
Heated Lid ON
Pause OFF
In denat 94ºC 5 min.
Hot start OFF
30 cycles
94 ºC 30 secs.
55ºC 30 secs.
70ºC 45 secs.
72ºC 5 min
hold 10ºC
