From 2009.igem.org
Wet
GVP Cluster
Transporters
GlpF
PCR 2 was add on gel again as noted below at accumulation.
PCR 1 was
GlpF PCR1
PCR1
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
|
|
|
|
A 2% agarose gel was used, a band of 105 bp was expected.
The band in row 2 was cut and purified using the zymo research Zymoclean TM Gel DNA Recovery Kit.
The purified PCR1 DNA and PCR2 DNA were used to run PCR3
GlpF PCR3
PCR1.1
Component | amount
|
2x Phusion MM
| 12.5 uL
|
GlpF PCR1
| 6 uL
|
GlpF PCR2
| 6 uL
|
|
|
GlpF PCR1 program
| Temperature
| Time
|
Denaturing
| 98°
| 2.00 min
|
Start Cycles 30X
|
Denaturing
| 98°
| 10 sec
|
Annealing
| 60°
| 10 sec
|
Elongation
| 72°
| 30 sec
|
| End cycles
|
Final elongation
| 72°
| 5 min
|
Hold
| 4°
| Forever
|
|
|
|
The results of the 1% agarose gel can be found at 28/07/09
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
- Restriction digest on pSB3K3-const. promoters and pSB1AC3-cons. promoters and pSB1A2-lac promoter
- Plasmids isolated with sigma plasmid isolation kit from o/n culture 24-25 July
- Restriction was done with 250-500ng plasmid
|
|
Check pLac insert
10x Tango buffer
| 2 uL
|
pSB1A2-lac1&2 (19-22 ng/uL)
| 17 uL
|
EaeI (CfrI)
| 1 uL
|
Total
| 20 uL
|
|
|
Check const. promoter insert (without promoter single cut, with 2x
10x Orange buffer
| 2 uL
|
pSB3K3-HML or pSB1AC3-HML
| 7 or 17 uL (depending on conc.)
|
StyI (Eco130I)
| 1 uL
|
MilliQ
| 0 or 13 uL
|
Total
| 20 uL
|
|
- Expected fragments are
- pSB1A2-Lac: 1400, 650, 180, 40bp
- pSB3K3-const. promoter: 2620, 160bp
- pSB3K3: 3100bp
- pSB1AC3-const. promoter: 1770, 1319bp
- pSB1AC3: 3400bp
- Loaded on a 1% Agarose gel (TBE)
- Contents are (from left to rigth):
1KB marker, pSB2K3-H, pSB2K3-M, pSB2K3-L, pSB2K3, pSB1AC3-H, pSB1AC3-M, pSB1AC3-L, pSB1AC3, pSB1A2-Lac1, pSB1A2-Lac2.
- The fragments seem to be on the correct higth, though there are also extra bands seen, which may be uncut plasmid DNA.
- O/n cultures of pBAD
- Pick 4 colonies of TOP10 + pSB1A2-pBAD/AraC
- Grow o/n in ~4ml LB-amp in the warm room.
Dry
Finished RPU computations!