From 2009.igem.org
Wet
GVP Cluster
Transporters
Metal Accumulation
Vectors
In order to check the VR VF2 primers that were ordered this year a gradient PCR was done with a vector that is expected to work (pSB3K3), a pUC vector and a negative control with no vector.
|
|
VR-VF2
Component | amount
|
MasterMix NH4
| 21 uL
|
VF2 iGEM09
| 1 uL
|
VR iGEM09
| 1 uL
|
vector
| 1 uL
|
Taq polymerase
| 1 uL
|
|
|
VR VF2 gradient program
Stage
| Temperature
| Time
|
Denaturing
| 95°
| 4 min
|
| Start Cycles 30X
|
Denaturing
| 95°
| 1.30 min
|
Annealing
| Gradient 50°-62°
| 30 sec
|
Elongation
| 72°
| 1.30 min
|
| End cycles
|
Final elongation
| 72°
| 6 min
|
Hold
| 4°
| Forever
|
|
As a check-up we also did a restrition analysis for the sPB3K3 vector with EcoR1 and Spe1
double digestion
10x fast digest buffer
| 2 uL
|
pSB3K3 (22,5 ngr/uL)
| 8 uL (=180ngr up to 1ugr is recommended at fermentas)
|
EcoR1
| 1 uL
|
Spe1
| 1 uL
|
MilliQ
| 8 uL
|
Total
| 20 uL
|
- Mix gently and spin down
- incubate at 37° for 30 min.
Results of the PCR and restiction shown below.
Dry