From 2009.igem.org
Wet
GVP Cluster
Transporters
GlpF
- PCR 2 was add on gel again as noted below at accumulation.
GlpF PCR1
PCR1.1
Component | amount
|
2x Phusion MM
| 12.5 uL
|
MQ
| 10.25 uL
|
GlpF Fw
| 1 uL
|
GlpF MutRev
| 1 uL
|
DNA GlpF Full Lenght (extracted as noted below at accumulation)(not added to tube nr2)
| 0.25 uL
|
|
|
GlpF PCR1 program
| Temperature
| Time
|
Denaturing
| 98°
| 2.00 min
|
Start Cycles 30X
|
Denaturing
| 98°
| 10 sec
|
Annealing
| 60°
| 10 sec
|
Elongation
| 72°
| 10 sec
|
| End cycles
|
Final elongation
| 72°
| 5 min
|
Hold
| 4°
| Forever
|
|
|
|
Metal Accumulation
Gel with ArsR PCR((24/07/09)
ArsR/GlpF gel extraction
- - Used zymo research Zymoclean TM Gel DNA Recovery Kit to extract ArsR, GlpF PCR2 and GlpF FullLenght from gel (cut from gel as shown in figure above).
GlpF PCR1
PCR Mix
12,5 uL
| Phusion
|
1 uL
| GlpF FW primer
|
1 uL
| GlpF RevMut primer
|
0,25 uL
| GlpF Full Lenght DNA (extracted as noted above)(not in tube2)
|
10,25 uL
| MQ
|
Vectors
Dry
Finished RPU computations!