Team:Groningen/Notebook/27 July 2009

From 2009.igem.org

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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 Forever

A 2% agarose gel was used, a band of 105 bp was expected.

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

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 Forever

The results of the 1% agarose gel can be found at 28/07/09

Metal Accumulation

Gel with ArsR PCR((24/07/09)

270709 ArsR+GlpF.JPG

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. F102471 2009-07-27 restriction2.JPG

    • 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!


April
MTWTFSS
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6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30
May
MTWTFSS
        1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30 31
June
MTWTFSS
1 2 3 4 5 6 7
8 9 10 11 12 13 14
15 16 17 18 19 20 21
22 23 24 25 26 27 28
29 30
July
MTWTFSS
    1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30 31
August
MTWTFSS
          1 2
3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
31
September
MTWTFSS
  1 2 3 4 5 6
7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30
October
MTWTFSS
      1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30 31
November
MTWTFSS
            1
2 3 4 5 6 7 8
9 10 11 12 13 14 15
16 17 18 19 20 21 22
23 24 25 26 27 28 29
30