From 2012.igem.org
Saturday, 23th June
Quick Change mutagenis to remove NgoMIV from pYES2
Investigator: Ingmar, Volker
Aim of the experiment: Generation of an RFC 25 compatible version of the pYes2 Vector.
PCR reaction batch
| volume
| reagent
|
| 2.5 µl
| 10x Pfu Ultra II buffer
|
| 4 µl
| Plasmid P7 pYes2_RFC25 MCS template
|
| 0.5 µl
| 1:10 dilution of O38 (10 pmol/µL)
|
| 0.5 µl
| 1:10 dilution of O39 ((10 pmol/µL)
|
| 17 µl
| ddH2O
|
| 0.5 µl
| dNTP mix
|
| 0.5 µl
| Pfu Turbo DNA polymerase (2.5 U / µl)
|
PCR cycling parameters
| Segment
| Cycles
| Temperature
| Time
|
| 1
| 1
| 95 °C
| 30 sec
|
| 2
| 15
| 95°C
| 30 sec
|
|
|
| 55°C
| 1 min
|
|
|
| 68°C
| 6 min
|
"
