interpretation:
bands had right size but pExStrepDigSplitFoka showed
unexpectedly low concentration
->gelextraction
ligation of
- pExHisFluaSplitFoki (vector) and StrepDigSplitFoka (insert)
- pExHisFluaSplitFoki (vector) and RBS_StrepDigSplitFoka (insert)
phage display
- OD600 of the pre-culture should be 0,2
- 37°C shaker
- After 70 minutes: Measure OD600 (should be about 0,6-0,7)
- Apportion culture into 10x50 ml Falkon tubes
- 15 minutes on ice
- Centrifuge 10 minutes at 4°C / 2500 g
- discard supernatant, wash pellet with 25 ml H2O (let dry upside down)
- resuspend pellets in 25 ml H2O (10-> 8 tubes)
- 15 minutes on ice
- Centrifuge 10 minutes at 4°C / 2500 g
- Discard supernatant, resuspend pellets in 25 ml H2O (8-> 4
tubes)
- 15 minutes on ice
- Centrifuge 10 min at 4°C / 2500
- Discard supernatant, resuspend pellets in 25 ml 10% DMSO (4->
2 tubes)
- 5 minutes on ice
- Centrifuge 10 minutes at 4°C / 2500 g
- Discard supernatant, resuspend pellets in 5 ml 10% DMSO (2 tubes)
- 5 minutes on ice
- Centrifuge 10 min at 4°C / 2500 g
- Discard supernatant, pool pellets in 1 ml 10% DMSO
- Measure OD600 (1:100 dilution), OD should be 0,4
- Make aliquots à 80 µl, souse with N2
- Store at -80°C
glycerol stock of pEx_HisFluASplitFoki in BL21de3
SDS gel of pool (elution fraction 2-5) from protein
expression (HisFluASplitFoki) from 01.10.09
--> do a Western Blot
preparative gel
[[Image:Freiburg09_021009_umklonierung.JPG|none|thumb|Agarose gel;
Lanes: pMASplitFoka, 2.pMAFoka, 3.pMAFoki, 4.pMASplitFoki,
5.pMALongLiFoka, 6.pMALongliFoki, 7.pMAShortli, 8.pMAMiddleli,
9.pMALongli, 10.pExStrepFluA<|400x400px]]
->interpretation: just the linker resulted in too short
fragments,
thus the hybridized linkers have to be cloned in pMA directly
gelextraction of digest from 02.10.09 and also
pMAShortliFoka, pMAShortliFoki, pMAMiddleliFoka, pMAMiddleliFoki from
01.10.09
03.10.09 Manu, Hannes, Gerrit, Caro, Timo, Christoph
plasmid prep of pET and pBad and Glytsocks(Xbl)
new aliquots Ampicilin (70%EtOH)
Test digest M13dsDNA+fokI as control for dsDNA , the
pictures
showed the two expected lanes(hardly visible on the printout).
[[Image:Freiburg09 091003 M13test 005.jpg|none|thumb|Agarose gel;
M13dsDNA test digest; Lanes: DNA ladder mix ,empty , M13dsDNA02.10.09,
M13dsDNA02.10.09 digest fokI |400x400px]]
digest of - pEx-Strep-Dig-LongLinker-Fok(inactive)
preparative agarose gel, lanes: 1. Genruler DNA ladder
mix of fermentas, 2. pBADvector, 3. pBADinsert/dummy, 4.pJS418
preparative agarose gel, lanes: 1. Genruler DNA ladder
mix of fermentas, 2. pJS419, 3. pExHisSplitFoka, 4. pExStrepDigSplitFoka
interpretation:
pExStrepDigSplitFoka seemed
to be a wrong construct, the other constructs showed bands of the right
size even if the concentration of the PCR pBAD insert seems to be very
low
His-tag purification of HisDigSplitFoka (periplasm export)
with Ni-NTA
column. Washing buffer: 25mM imidazole
-cells were sonicated for 2 x 1min before filtering with
0.45µm and 0.22µm filter
Ligation
- Dephphorylation of pBAD Gelex
- 1µl Fast AP
- 5.5µl fast AP buffer
-1.5µl water
--> Solution was given to eluat
- for each ligation:
- 6µl Insert
-3µl Vetor
-1µl Quickligase
-10µl buffer
--> pBAD
Inoculation
of ER2738 M13: 250ml Erlenmeyerkolben+ 50ml LB+TET in 37°c room
overnight
Yesterdays improved pcr resulted in a lot of product, but
still
unspecific ones.. made new one with even higher annealing temperature
(69°C) and a bit less cycles (35)
New AGO M13 ssDNA assay using 5`-phosphorylated Oligos A1
and A4 _see
gele_
no differend results were obtained compaired to the assays using
unphorphorylated oligos
Started dialysis to transfer the leftover AGO-proteins into
the assay
buffer
SDS gel of protein purification of HisDigSplitFoka
(periplasm)
from 06.10.09
[[Image:Freiburg09_071009_pg_DsbAHisDigSplitFokA_28grad006.jpg|none|thumb|SDS
gel, pEx_DsbA_HisDigSplitFoka, lanes: NEB prestained protein marker
broad range, Elution fraction 1, Elution fraction 2, Elution fraction
3, Elution fraction 4, Elution fraction 4, Elution fraction 5, Elution
fraction 6, flow through fraction 3, washing fraction 2, periplasm
extract (frozen over night at -80°C)|400x400px]]
Inoculation 4 Clones respectively:
-
pMA-His-Dig-MiddleLinker-Foki in XLBlue
- pJS419-HIs-Dig-Split-Foka- in XLBlue
- pJS418-HIs-DIg-Split-Foka- in XLBlue
- pEX-Strep-FluA-MiddleLinker-Foki in XL1blue rest
- pEX-Strep-FluA-LongLinker-Foki in XL1blue rest
- pMA-CAT in XLblue 10µl
-pEX-Strep-FluA-MiddleLinker-FokA in XL1blue rest
2 Clones respectively:
-pEX-His-FluA-Split-Foki + Glycerolstock vom 26.08.09
- pEX-Strep-Dig-Split-Foka + Glycerolstock vom 29.08.09
Starter culture of pEx-DsbAHisDigSplitFoka in
Bl21de3
two step PCR assembly of DsbA, His_Fos, and SplitFoka
- program name: Assembly
- three different samples:
1. without DMSO, 2. with DMSO, 3. without
DMSO and with last primers just added after first step
preparative gel of the PCR samples
-> primer haven't been diluted an probably made all secondary
structures, has to be repeated
digest of pBAD with AgeI and new PCR with insert digested
with XmaI
preparative gel with digest and
test digest of pJS419_HisDigSplitFoka and
pJS419_StrepDigSplitFoka
DNA: 5µl
Enzymes: 0.5µl
of BamHI and MfeI each
Buffer: 1µl buffer 4
BSA (10fold): 1µl
Water: 2µl
-> pJS419_HisDigSplitFoka showed bands of the right size and was
sequenced with primer sf_lac P1
plasmidpreparation of - pEXHisFluASplitFoki
- pExStrepDigSplitFoka
-> low concentrations
digest of pExHisFluASplitFoki, pExStrepDigSplitFoka and
RBS_StrepDigSplitFoka
pExHisFluASplitFoki: 15µl
Enzymes: 1µl of
PstI and 1.5µl SpeI
Buffer: 3µl buffer 2
BSA (100fold): 0.5µl
Water: 9µl
pExStrepDigSplitFoka: 15µl
Enzymes: 1µl of XbaI and 1.5µl PstI
Buffer: 3µl buffer 3
BSA (100fold): 0.5µl
Water: 9µl
pExStrepDigSplitFoka: 10µl
Enzymes: 1µl of XbaI and 1.5µl PstI
Buffer: 3µl buffer 3
BSA (100fold): 0.5µl
Water: 14µl
Gelextraction of digest
1) Insert: pEX-Strep-Dig-Split-Foka( digested with xba and pst)
2)Vector:
pEX-His-Flua-Split-Foki (digested with spe, pst)
3)PCR Product: RBS-Strep-Dig-Split-FokA(digested with pst, xba)
Stardet production of Phages baering the phagmid vektors
with
pJS_TorA-flag-AGO-noAmber-CDg3p and the pJS_TorA-flag-AGO-CDg3p, 480 ml
each. see phageproduction protocoll day 1
prepaired ELISA with anti-flag antibodies
made electro competent cells for transformation with the
ago
phagmidbibliothek
08.10.09 Manu, Christoph, Julia, Caro, Timo, Hannes
Poured SDS-Gels
Phage ss DNA -Over night cultured ER2738 in 100ml LB+Tet
dilute on
OD600=0,4 in 50ml LB+Tet
-Transfomation with 3µl M13 Phage stock (-80°) at
10:30Uhr , incubated for 4-5 hours at 37°C in shaker
-Centrifuge in 50ml Falcon tube at 5000rpm for 20 min, 4°C
-Phages are in the supernatant, add 1/7 PEG/NaCl (for 50ml culture 7ml
precipitation over night on ice in -4°C room
Miniprep
- 2 clones respectively:
-A = pEX strep dig split
foka
Finished Phage production(see protocol day 2): We obtained
approximately 3.8
10^12 pJS_TorA-flag-AGO-noAmber-CDg3p (449) and 1.1
10^13 pJS_TorA-flag-AGO-CDg3p (448) -> aplyed all of
them to
the anti-Flag ELISA
Anti-Flag ELISA was successfull with a slight but detctable
signal via
anti M13 VCS Antibodies (with peroxidase):
see
405 nm absorption in wells G9-10 (448) and G7-8 (449) compaired to
positive control (G5-6) and negative control (G3-4 and D3-10) detected
about half an hour after the ABTS substrat was addet.
1
2
3
4
5
6
7
8
9
10
11
12
A
0.0470
0.0420
0.0440
0.0430
0.0480
0.0510
0.0460
0.0470
0.0470
0.0480
0.0450
0.0460
B
0.0470
0.0460
0.0480
0.0480
0.0460
0.0450
0.0470
0.0560
0.0470
0.0450
0.0450
0.0440
C
0.0460
0.0470
0.0500
0.0450
0.0430
0.0460
0.0450
0.0460
0.0430
0.0420
0.0440
0.0470
D
0.0490
0.0470
0.1590
0.1610
0.1920
0.1720
0.2210
0.2500
0.2130
0.2680
0.0460
0.0480
E
0.0490
0.0490
0.0450
0.0490
0.0430
0.0490
0.0460
0.0430
0.0460
0.0420
0.0450
0.0440
F
0.0490
0.0430
0.0470
0.0440
0.0470
0.0470
0.0440
0.0450
0.0500
0.0460
0.0450
0.0450
G
0.0500
0.0440
0.2530
0.2510
3.9180
3.8800
0.9560
0.9380
1.2420
1.2360
0.0440
0.0460
H
0.0480
0.0430
0.0510
0.0470
0.0480
0.0460
0.0470
0.0450
0.0450
0.0440
0.0460
0.0460
Made new PCR for ssDNA from pET39b+ fragment and
gained approximately
200 ng of ssDNA after PCR and gelextraction
digest of pExHisFluASplitFoki, pExStrepDigSplitFoka and
RBS_StrepDigSplitFoka
pExHisFluASplitFoki: 15µl
Enzymes: 1µl of PstI and 1.5µl SpeI
Buffer: 3µl buffer 2
BSA (100fold): 0.5µl
Water: 9µl
pExHisDigSplitFoka: 15µl
Enzymes: 1µl of XbaI and 1.5µl PstI
Buffer: 3µl buffer 3
BSA (100fold): 0.5µl
Water: 14µl
RBSStrepDigSplitFoka: 5µl
Enzymes: 1µl of XbaI and 1.5µl PstI
Buffer: 3µl buffer 3
BSA (100fold): 0.5µl
Water: 19µl
->made two digest of PCR construct
Gelextraction of digest
1) Insert: His-Dig-Split-Foka( digested with xba and pst)
2)Vector: pEX-His-Flua-Split-Foki (digested with spe, pst)
3)PCR Product: RBS-Strep-Dig-Split-FokA(digested with pst, xba)
and also PCR purification of RBS-Strep-Dig-Split-FokA
preparative gels of digests
->see picture
ligation of
-pEX-His-Flua-Split-Foki_His-Dig-Split-Foka
inoculation of pJS419_HisDigSplitFoka in LB +
chloramphenicol
new two step PCR with all three plasmids for Fos construct
(pMADsbA, pMAFos, pMASplitFoka, this time with right primer dilution
and preparative agarose gel
->interpretation: no band of 921bp was visible
one step PCR of pMADsbA, pMAFos, pMASplitFoka separated and
preparative agarose gel
->see picture
->interpretation: pMAFos and pMASplitFoka showed bands of the
right
size (230bp and 657bp respectively), of pMADsbA no product was visible
new one step PCR of pMADsba with newly prepared 1:1000
dilution
test digest of plasmidpreparations from today
->see picture
-Over night in 4°C room precipitated phages: centrifuged for 20
min at 5000rpm and 4°C
-Discard supernatant, resuspended pellet in 2ml TBS (no pellet
recognized)
-Separated solution in 2 Eppendorf tubes, centrifuged for 10 min at
13000rpm
-Decant the supernatant into new eppis, precipitate with
170µl PEG/NaCl and leave for 1 hour on ice
Miniprep
- pJS 419-his-dig-split-foka (clon 1+2)
- Glycerolstock of clon1 and 2
- 300µl Glycerol
- 700 µl culture
- Pellets from clones 3,5,6
Nanodrop data:
pJS 419-his-dig-split-foka-clon1= 509.5 ng/µl
pJS 419-his-dig-split-foka-clon2 =468 ng/µl
Inoculation of ER2738 in 2x 2L conical flask with 1L LB+1ml
Tet, over
night in 37°C room
digest for recloning of pMA constructs:
pMAShortlinkerFoka clone 1, prep from 03.10.09, pMAMiddlelinkerFoka
clone 2, prep from 03.10.09, pMALonglinkerFoka clone 1, prep from
10.09.09, pExDsbAHisDigSplitFoka, clone 1 from 02.10.09,
pExDsbAStrepDigSplitFoka, clone 1 from 02.10.09: 10µl
Enzymes: 1µl of NgoMIVI and 1.5µl PstI
Buffer: 3µl buffer 1
BSA (10fold): 3µl
Water: 10,5µl
pMAHisDig clone 2, prep from 24.08.09, pMAStrepDig clone 2, prep from
25.09., pExStrepDig clone 2, prep from 25.09.09, pMABB057 from
01.10.09: 10µl each
Enzymes: 1µl of AgeI and 1.5µl PstI
Buffer: 3µl buffer 1
BSA (10fold): 3µl
Water: 10,5µl
PCR assembly with pMADsbA, different approaches (Taq
Polymerase or
Phusion Polymerase, differnt Tm) and combination PCR with pMADsba and
pMAFos together
analytical gel of PCRs -> didn't work
preparative gels of digests
-> see picture
-> no inserts with pExDSba_Foka constructs because they have no
NgoMIV site any more
Starter culture of pEx_DsbA_StrepDigSplitFoka
(Bl21de3)
plasmid prep of pEx_DsbA_StrepDigSplitFoka
plasmid prep of pEx_HisDigSplitFoka
M13 ssDNA produced with bacterial of Julia and
tried another
variance with 100ml+tet(1:1000)+ER2738 grow to OD 0,2 then infected
withM13phage particles and let it grow for 2h. After this followed the
qiagen M13 protocol for M13Dna
Made new BB-AGO PCR using digested AGO Gene without the
vector-> still no expected bands to be seen...
Made new PCR to generate more pET39b+ ssDNA because
yesterdays had
insufficient concentration for the AGO cleavage assay
Made digest of errorprone PCR product of the AGO-G3P
constructs
(from 02.10.; one with, one without Amber) via NheI and SfiI to gain
new Phagmid library
13.10.09, Laura, Caro, Christoph,Manu, Hannes, Julia
Miniprep
-pMA-fos 1
-pMA-fos 2
-pMA his flua split fokI
- pMA strep dig split foka
-pMA long linker
testdigest of Plasmidprep from today
15 µl for 6µl loading dye
-buffer 2 1µl
-xbaI 0.5µl
-pstI 0.75µl
-DNA 2 µl
-water 9.75µl
-BSA 1µl
--> Gelbild
- Glycerolstocks are in in box of 5/10/2009
made electrocompetent XL1 Blue cotransformed cells
(pExHisFluASplitFoki and pJS419HisDigSplitFoka)
->stored in -80°C
culture of ER2738 in LB Tet for cotransformation in
afternoon
test in vivo assay - electroporation with new
electrocompetent cells
and M13 ssDNA at 1.7kV
- 1.5h on 37°C shaker at 750rpm
- precipitation of phages
- infection of ER2738 with different dilutions of phages
- mixed cells with Top agar and plated them on IPTG/XGAL plates
-> 37° shaker
inoculation of
-pMASplitFoka clone 1 from prep 04.10.09
-pMAStrepDig clone 2 from prep 25.09.09
-pMAMiddleFoka clone 2 from prep 04.10.09
-pMAShortFoka clone 2 from prep 04.10.09
-pMAFoka clone 1 from prep 04.10.09
-pExDsbAStrepDigSplitFoka clone 1 from prep 02.10.09
-pMALongFoka clone1 prep from 10.09.09
-pMAFos 13.10.09
-pBAD_CAT 13.10.09
-pBAD_Foka 13.10.09
-pMADsbAHisDigSplitFoka 13.10.09
-pMADsbAStrepDigSplitFoka 13.10.09
-pExFosSplitFoka 13.10.09
-pMAFosSplitFoka 13.10.09
protein purification of DsbA_StrepDigSplitFoka with Strep
column
digestion, 1% agarose gel and gel extraction of
- pMA-LongLinker-FokA (10.09., clone 1) with EcoRI + SpeI
- pMA-His-FluA-Split-FokI (13.10.) with SpeI + PstI
- pMA-SplitLinker-FokA (04.10., clone 2) with NgoMIV + PstI =>
wasn't ok on the gel, inoculated clone 1
- pMA-ShortLinker-FokA (04.10., clone 2) with NgoMIV + PstI
- pMA-MiddleLinker-FokA (04.10., clone 1) with NgoMIV + PstI
- pMA-His-Dig (24.08., clone 2) with AgeI + PstI
- pMA-Strep-Dig (25.09., clone 2) with AgeI + PstI
- pEx-Strep-FluA-MiddleLinker-FokI (08.10., clone 1) with XbaI + AgeI
- pEx-Strep-FluA-MiddleLinker-FokA (08.10., clone 1) with XbaI + AgeI
- pEx-Strep-FluA-LongLinker-FokI (08.10., Klon 1) with XbaI + AgeI
- pMA (01.10., c=500ng/µl) with XbaI + AgeI
- pMA (01.10., c=500ng/µl) with EcoRI + SpeI
did transformations of the ligations in RV308, plated on
LB-Amp + 1%
Gluc
ELISA -Blocking
-Washing with TBST-EDTA 5x
-Loaded immunotubes (1.5ml) and wells 100µl each with 0.5mM
biotin-target DNA
-Incubation at 20°C in shaking
-Incubatet Phages with Oligo for 30min at 55°c waterbath
-Washing of wells and immunotubes with TBST-EDTA 5x
-Loaded 2 wells+controls with 449 phages+Oligo, 1+control with P1
phage+Oligo and 1+control as a control
-Incubation in shaker at 20°C
-Loaded immunotubes (each 1.5 ml) with 50µl P1,
25µl 449Phage+ Oligo in TBST-EDTA
-Incubation in shaker at 20°C
-Washing of wells and immunotubes with TBST-EDTA 5x
-Loaded 100µl/well anti-M13 ab(1:1000), incubated for 1 hour
at 20°C in shaker
-Immunotubes for panning: 1)2x 1ml TBS+MnCl2(5mM in 5ml), shaker for 20
min, decanted supernatant into eppis, washed with TBS 3x
-2)2x 1ml TBS-T+MnCl2 (5mM in 5ml), shaker for 20 min, decanted
supernatant into eppis, washed with TBS-T 3x
-3)2x 1ml DNAse shaker for 20 min, decanted supernatant into eppis
Made new "AGO BB PCR" this time just using the
ad-on-tail-primer
for the bb pre- and suffix, so that hte EcoRI site should remain. Still
the same not fitting bands were obtained-> the problem might be
caused by multimerising of the primers, even so we were not able to
predict any of tose using vectir nti or compairable programms...
AGO assay using different targets-> were not able to
reproduce the presumed cutting event from Saturdays assay probably due
to the lower DNA concentration
Made ELISA with strep-biotinylated target oligo coated
surface-> no binding of AGO bearing phages was detected using
anti-M13 HRP and ABTS
Made test panning just like the ELISA above and eludet with
TBS-MnCl2
(5mM), then TBST-MnCl2 (5mM) and then with DNase I
"
October 
