Team:Groningen/Notebook/27 July 2009
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===Transporters=== | ===Transporters=== | ||
+ | GlpF | ||
+ | PCR 2 was add on gel again as noted below at accumulation. | ||
+ | |||
+ | PCR 1 was | ||
+ | '''GlpF PCR1''' | ||
+ | {| | ||
+ | | | ||
+ | <!--Tabel 1 hier--> | ||
+ | |||
+ | {| border="1" | ||
+ | |+ '''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 | ||
+ | |} | ||
+ | |||
+ | |width="10"| | ||
+ | | | ||
+ | <!--Tabel 2 hier--> | ||
+ | {| | ||
+ | ! 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 | ||
+ | |} | ||
+ | |width="10"| | ||
+ | | | ||
+ | |} | ||
+ | |||
+ | A 2% agarose gel was used, a band of 105 bp was expected. | ||
+ | |||
+ | [[Image:F102471_2009-07-27_16hr_21min_GlpFPCR1_noted.JPG]] | ||
+ | |||
+ | 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''' | ||
+ | {| | ||
+ | | | ||
+ | <!--Tabel 1 hier--> | ||
+ | |||
+ | {| border="1" | ||
+ | |+ '''PCR1.1''' | ||
+ | ! Component !! amount | ||
+ | |- | ||
+ | ! 2x Phusion MM | ||
+ | | 12.5 uL | ||
+ | |- | ||
+ | ! GlpF PCR1 | ||
+ | | 6 uL | ||
+ | |- | ||
+ | ! GlpF PCR2 | ||
+ | | 6 uL | ||
+ | |} | ||
+ | |||
+ | |width="10"| | ||
+ | | | ||
+ | <!--Tabel 2 hier--> | ||
+ | {| | ||
+ | ! 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 | ||
+ | |} | ||
+ | |width="10"| | ||
+ | | | ||
+ | |} | ||
+ | The results of the 1% agarose gel can be found at 28/07/09 | ||
===Metal Accumulation=== | ===Metal Accumulation=== | ||
Line 40: | Line 179: | ||
===Vectors=== | ===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 | ||
+ | |||
+ | {| | ||
+ | | | ||
+ | <!--Tabel 1 hier--> | ||
+ | |width="10"| | ||
+ | | | ||
+ | |||
+ | {| border="1" | ||
+ | '''Check pLac insert''' | ||
+ | |||
+ | |10x Tango buffer | ||
+ | |2 uL | ||
+ | |- | ||
+ | |pSB1A2-lac1&2 (19-22 ng/uL) | ||
+ | |17 uL | ||
+ | |- | ||
+ | |EaeI (CfrI) | ||
+ | |1 uL | ||
+ | |- | ||
+ | |Total | ||
+ | |20 uL | ||
+ | |- | ||
+ | |} | ||
+ | |||
+ | <!--Tabel 2 hier--> | ||
+ | |width="10"| | ||
+ | | | ||
+ | |||
+ | {| border="1" | ||
+ | '''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. | ||
+ | [[Image:F102471 2009-07-27 restriction2.JPG|250px]] | ||
+ | **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== | ==Dry== | ||
+ | Finished [[Team:Groningen/Promoters|RPU computations]]! | ||
{{Team:Groningen/Notebook/Day/Footer}} | {{Team:Groningen/Notebook/Day/Footer}} |
Latest revision as of 09:56, 31 July 2009
Wet
GVP Cluster
Transporters
GlpF PCR 2 was add on gel again as noted below at accumulation.
PCR 1 was GlpF PCR1
|
|
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
|
|
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
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
|
|
- 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):
- Expected fragments are
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!
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