Team:Freiburg bioware/Notebook/October

   FREiGEM  

  Home   The Team </a> <ul> Overview</a></li> Portraits</a></li> </ul> </li> <span class="l"> <span class="t">The Project </a> <ul> Summary</a> </li> Subprojects</a></li> Highlights</a></li> </ul> </li> <span class="l"> <span class="t">Human Practice </a> <ul> Ethics</a> </li> <li><a href="http://2009.igem.org/Team:Freiburg_bioware/Human_Practice/Safety">Safety</a></li> </ul> </li> <li><a href="http://2009.igem.org/Team:Freiburg_bioware/Notebook" class="active"> <span class="r"> Notebook </a></li> <li><a href="http://2009.igem.org/Team:Freiburg_bioware/cloning1"><span class="l"> <span class="t">Parts </a> <ul> <li><a href="http://2009.igem.org/Team:Freiburg_bioware/cloning1">Basic Parts</a></li> <li><a href="http://2009.igem.org/Team:Freiburg_bioware/cloning">Composite Parts</a></li> </ul> </li> <li><a href="http://2009.igem.org/Team:Freiburg_bioware/Collaboration"><span class="l"> <span class="t">Collaboration </a></li> <li><a href="http://2009.igem.org/Team:Freiburg_bioware/Modeling"><span class="l"> <span class="t">Modeling </a></li> </ul> <h2 class="art-PostHeaderIcon-wrapper"> <img style="width: 28px; height: 25px;" alt="" src="http://2009.igem.org/wiki/images/2/2a/Freiburg09_Post_tanne_2.png" /> October <h3 style="margin-left: 40px;">01.10.09, Hannes, Manu, Laura, Gerrit, Sarah, Christoph, Caro, Julia, Anika, Isabel

<ul style="margin-left: 40px;"> <li> Tetracycline; 10 x 1ml, Concentration: 25 g/l, stored in -20&deg;C, box with antibiotics </li> <li> Chloramphenicol; 10 x 1ml, Concentration: 25 g/l, stored in -20&deg;C, box with antibiotics </li> <li> plasmid preparation of pEx-His-FluA-Split-Fok(inactive)-RBS-Strep-Dig-Split-Fok(active) Klon 4 </li> <li> plasmid preparation of pEX-Strep-Dig-Split-Fok(active) Klon 1 </li> <li> glycerin stock of pEx-His-FluA-Split-Fok(inactive)-RBS-Strep-Dig-Split-Fok(active) Klon 4 (RV308), stored at -80&deg;C </li> </ul>

<ul style="margin-left: 40px;"> <li>protein purification of HisFluASplitFoki (expressed in BL21de3) with NiNTA column </li> <li>SDS gel of protein purification HisFluASplitFoki </li> <li>pooled fraction 2-5, dialysis over night in dialysis buffer (30mM NaCl, 20mM Tris-HCl, pH 7.4) </li> <li>phage display: - desalt ligation products (for electroporation)</li> </ul> pool samples (~80&micro;l 445+87/89 and ~52&micro;l 445+87/88)

add 1 volume of isopropanol, mix

-80&deg;C for 10 minutes

centrifuge for 10 minutes at max rpm, 4&deg;C; discard supernatant

add 1 volume of 75% ethanol (without mixing)

centrifuge for 10 minutes at max rpm, 4&deg;C; discard supernatant

let dry on heat block (50&deg;C), lid open

add 25&micro;l water

thermo shaker for 1h, 45&deg;C, 1000rpm - PCR (4 samples each template):

buffer (with MgCl2): 5&micro;l

primer #7: 1,5&micro;l

primer #1: 1,17&micro;l

dNTPs: 2&micro;l

Taq: 1&micro;l

MnCl2: 0,5&micro;l

MgCl2: 5&micro;l

water: 33&micro;l

DNA (425 or 428): 5&micro;l

- PCR:

DNA templates: pJs#448 (0,3&micro;l), pJs#449 (0,3&micro;l), pJs#375 (1&micro;l), pJs#413 (0,3&micro;l), pJs#445 (0,3&micro;l)

primers (#95, #7): 1,5&micro;l

high fidelity buffer: 5&micro;l

dNTPs: 1&micro;l

TMenzyme: 0,3&micro;l

water: 39,7&micro;l

<ul style="margin-left: 40px;"> <li> Overnight Culture RV308 for Competent Cells, on shaker in 37 &deg;C room </li> <li>analysis of sequences from 28.09.09 </li> <li>digest of pExStrepDigSplitFoka prep from 30.09.09 Plasmid: 15 &micro;l</li> </ul> water: 9 &micro;l

BSA: 0.5 &micro;l NEB iGEM stock

buffer: 3 &micro;l buffer 3 NEB iGEM stock

Restriction_enzyme_1 : 1,5 &micro;l XbaI from NEB KuKlabstock

Restriction_enzyme_2 : 1 &micro;l PstI from NEB KuKlabstock

<ul style="margin-left: 40px;"> <li>digest of PCR product RBSStrepDigSplitFoka from 30.09.09 Plasmid: 10 &micro;l </li> </ul> water: 14 &micro;l

BSA: 0.5 &micro;l NEB iGEM stock

buffer: 3 &micro;l buffer 3 NEB iGEM stock

Restriction_enzyme_1 : 1,5 &micro;l XbaI from NEB KuKlabstock

Restriction_enzyme_2 : 1 &micro;l PstI from NEB KuKlabstock

pExHisFluaSplitFoki prep from 24.08.09 Plasmid: 15 &micro;l
 * digest of

water: 9 &micro;l

BSA: 0.5 &micro;l NEB iGEM stock

buffer: 3 &micro;l buffer 2 NEB iGEM stock

Restriction_enzyme_1 : 1,5 &micro;l XbaI from NEB KuKlabstock

Restriction_enzyme_2 : 1 &micro;l SpeI from NEB KuKlabstock

-&gt; put in 37&deg;C room for 2h -&gt;preparative gel

interpretation: bands had right size but pExStrepDigSplitFoka showed unexpectedly low concentration

-&gt;gelextraction

ligation of

- pExHisFluaSplitFoki (vector) and StrepDigSplitFoka (insert)

- pExHisFluaSplitFoki (vector) and RBS_StrepDigSplitFoka (insert)

<ul style="margin-left: 40px;"> <li>Inoculation of - pJS419_StrepDigSplitFoka</li> </ul> - pJS419_HisDigSplitFoka

- pEx_HisFluA

- pEx_HisDigMiddleLiFoka

<ul style="margin-left: 40px;"> <li>digest pMA_ShortlinkerFoki/a, pMA_MiddleLinkerFoki/a and pMA_scFa-anti_NIP for recloning </li> <li>Inoculation of pEx_HisFluASplitFoki in BL21de3 with colony of transformation from 28.09.09 --&gt; make glycerol stocks tomorrow </li> <li>agarose gel (1%) of the digest of pMA_ShortlinkerFoki/a, pMA_MiddleLinkerFoki/a and pMA_scFa-anti_NIP for recloning </li> <li>cut out insert bands (digestion of pEX didn't work, because there's no EcoRI recognition site any more) </li> <li>gel slices stored at -4&deg;C over night </li> </ul> <h3 style="margin-left: 40px;">02.10.09 Laura, Manu, Anika, Julia, Hannes, Gerrit, Isabell, Sarah, Caro, Timo, Christoph, Max

<ul style="margin-left: 40px;"> <li>phage display - OD600 of the pre-culture should be 0,2 </li> </ul> - 37&deg;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&deg;C / 2500 g

- discard supernatant, wash pellet with 25 ml H2O (let dry upside down)

- resuspend pellets in 25 ml H2O (10-&gt; 8 tubes)

- 15 minutes on ice

- Centrifuge 10 minutes at 4&deg;C / 2500 g

- Discard supernatant, resuspend pellets in 25 ml H2O (8-&gt; 4 tubes)

- 15 minutes on ice

- Centrifuge 10 min at 4&deg;C / 2500

- Discard supernatant, resuspend pellets in 25 ml 10% DMSO (4-&gt; 2 tubes)

- 5 minutes on ice

- Centrifuge 10 minutes at 4&deg;C / 2500 g

- Discard supernatant, resuspend pellets in 5 ml 10% DMSO (2 tubes)

- 5 minutes on ice

- Centrifuge 10 min at 4&deg;C / 2500 g

- Discard supernatant, pool pellets in 1 ml 10% DMSO

- Measure OD600 (1:100 dilution), OD should be 0,4

- Make aliquots &agrave; 80 &micro;l, souse with N2

- Store at -80&deg;C

<ul style="margin-left: 40px;"> <li>glycerol stock of pEx_HisFluASplitFoki in BL21de3 </li> <li>SDS gel of pool (elution fraction 2-5) from protein expression (HisFluASplitFoki) from 01.10.09 --&gt; do a Western Blot </li> <li>new ampicilin 100 aliquots </li> <li>preparation of M13dsDNA </li> <li>chem. competent cells aliqots </li> <li>Periplasma Project Digestion:</li> </ul> - digested with xba and spe --&gt; GelBilder

<ul style="margin-left: 40px;"> <li> Gelextraction Min Elute Gel Extraction Kit</li> </ul> - mesure mass: StrepFokA = 50 mg<be> - JS418= 90mg</be>

<be>-JS419=80mg</be>

<be>- HisFokA = 250mg</be>

<be style="color: rgb(255, 0, 0);">Nandropdata</be>

<be></be> <be></be>

<be></be> <ul style="margin-left: 40px;"> <li><be>Ligation - with Quickligase JS119-StrepFokA</be></li> </ul> <be>JS118-Strep</be>

<be>JS118-HisFokA</be>

<be>JS19-HisFokA</be>

<be>- 15 min at 25&deg;C </be>

<be></be> <ul style="margin-left: 40px;"> <li><be>Transformation - 2YT-Medium 950 &micro;L to 50&micro;l cells and DNA </be></li> </ul> <be>BL21-JS190</be>

<be>BL21-JS119StrepFokA</be>

<be>BL21-JS118Strep</be>

<be>BL21-JS118HisFokA</be>

<be>BL21-JS19HisFokA - Plated on KM plats</be>

<be></be> <be></be>

<be></be> <ul style="margin-left: 40px;"> <li><be> Preparation of overnight cultures 5 ml LB medium each - pBad+Kan</be></li> </ul> <be>-pET39B(+)</be>

<be></be> <ul style="margin-left: 40px;"> <li><be>Amp</be></li> </ul> <be>--&gt; Stored in 37&deg;C room </be>

<be></be> <ul style="margin-left: 40px;"> <li><be>Digest: - pMASplitFoka</be></li> </ul> <be>- pMAFoka</be>

<be>- pMAFoki</be>

<be>- pMASplitFoki</be>

<be>- pMALongLiFoka</be>

<be>- pMALongliFoki</be>

<be>- pMAShortli</be>

<be>- pMAMiddleli</be>

<be>- pMALongli</be>

<be>- pExStrepFluA</be>

<be></be> <be></be>

<be></be> <ul style="margin-left: 40px;"> <li><be>preparative gel -&gt;interpretation: just the linker resulted in too short fragments, thus the hybridized linkers have to be cloned in pMA directly </be></li> <li><be>gelextraction of digest from 02.10.09 and also pMAShortliFoka, pMAShortliFoki, pMAMiddleliFoka, pMAMiddleliFoki from 01.10.09   </be></li> <li><be>digest and ligation into new pMA:</be></li> </ul> <be>-pMASplitFokA clone1 19.08.09</be>

<be>-pMAFokA clone2 05.08.09</be>

<be>-pMAFoki clone2 05.08.09</be>

<be>-pMASplitFoki clone2 06.08.09</be>

<be>-pMAshortLi clone2 10.09.09</be>

<be>-pMAmiddelLi clone2 10.09.09</be>

<be>-pMAlongLi clone1 10.09.09</be>

<be>-pMAlongLiFokA clone1 10.09.09</be>

<be>-pMAlongLiFoki clone1 10.09.09</be>

<be>digestion with EcoRI and SpeI (vector and insert) </be>

<be></be> <be></be>

<be></be> <ul style="margin-left: 40px;"> <li><be> Transformation of:</be></li> </ul> <be>-pMAFokA clone2 05.08.09</be>

<be> -pMAFoki clone2 05.08.09</be>

<be></be><be>-pMASplitFoki clone2 06.08.09</be>

<be></be><be></be>

<be></be> <ul style="margin-left: 40px;"> <li><be>plasmidprep of:</be></li> </ul> <be>1. pEX_DsbA+Strep+Dig+Split+FokA Clone1 Quick</be>

<be>2. pEX_DsbA+Strep+Dig+Split+FokA Clone2 Quick</be>

<be>3. pEX_DsbA+Strep+Dig+Split+FokA Clone3 Quick</be>

<be></be>

<be>4. pEX_DsbA+His+Dig+Split+FokA Clone1 Quick </be>

<be>5. pEX_DsbA+His+Dig+Split+FokA Clone2 Quick</be>

<be>6. pEX_DsbA+His+Dig+Split+FokA Clone3 Quick</be>

<be></be>

<be>7. pEX_DsbA+Strep+Dig+Split+FokA Clone1 --&gt; seq 02.10.09 Gerrit1</be>

<be>8. pEX_DsbA+Strep+Dig+Split+FokA Clone2 --&gt; seq 02.10.09 Gerrit2</be>

<be>9. pEX_DsbA+Strep+Dig+Split+FokA Clone3</be>

<be></be>

<be>10. pEX_DsbA+His+Dig+Split+FokA Clone1 --&gt; seq 02.10.09 Gerrit3</be>

<be>11. pEX_DsbA+His+Dig+Split+FokA Clone2 --&gt; seq 02.10.09 Gerrit4</be>

<be>12. pEX_DsbA+His+Dig+Split+FokA Clone3</be>

<be></be><be></be>

<be></be>

<be></be> <ul style="margin-left: 40px;"> <li><be>testdigest of: V1. pEX_DsbA+Strep+Dig+Split+FokA Clone1 Quick</be></li> </ul> <be>V2. pEX_DsbA+Strep+Dig+Split+FokA Clone2 Quick</be>

<be>V3. pEX_DsbA+Strep+Dig+Split+FokA Clone3 Quick</be>

<be></be>

<be>V4. pEX_DsbA+His+Dig+Split+FokA Clone1 Quick </be>

<be>V5. pEX_DsbA+His+Dig+Split+FokA Clone2 Quick</be>

<be>V6. pEX_DsbA+His+Dig+Split+FokA Clone3 Quick</be>

<be></be>

<be>V7. pEX_DsbA+Strep+Dig+Split+FokA Clone1 </be>

<be>V8. pEX_DsbA+Strep+Dig+Split+FokA Clone2 </be>

<be>V9. pEX_DsbA+Strep+Dig+Split+FokA Clone3</be>

<be></be>

<be>V10. pEX_DsbA+His+Dig+Split+FokA Clone1 </be>

<be>V11. pEX_DsbA+His+Dig+Split+FokA Clone2 </be>

<be>V12. pEX_DsbA+His+Dig+Split+FokA Clone3</be>

<be></be>

<be>V13. pEX_Strep+Dig+Split+FokA prep from 25.09.09 (tobi stock)</be>

<be>V14. pEX_His+Dig+Split+FokA prep from 25.09.09 (tobi stock)</be>

<be>with NcoI-HF and XbaI 1h at 37&deg;C</be>

<be> --&gt; geldbild </be> <h3 style="margin-left: 40px;">03.10.09 Manu, Hannes, Gerrit, Caro, Timo, Christoph

<ul style="margin-left: 40px;"> <li>plasmid prep of pET and pBad and Glytsocks(Xbl) </li> <li>new aliquots Ampicilin (70%EtOH) </li> <li>Test digest M13dsDNA+fokI as control for dsDNA, the pictures showed the two expected lanes(hardly visible on the printout). </li> </ul> <ul style="margin-left: 40px;"> <li>digest of - pEx-Strep-Dig-LongLinker-Fok(inactive)</li> </ul> - pEx-Strep-Dig-MiddleLinker-Fok(inactive)

- pEx-Strep-Dig-ShortLinker-Fok(inactive)

- pEx-Strep-Dig-SplitLinker-Fok(inactive)

- pEx-His-Dig-SplitLinker_Fok(inactive)

- pEx-His-Dig-SplitLinker_Fok(active)

- pEx-Strep-Dig-SplitLinker-Fok(active)

- pEx-His-Dig-LongLinker-Fok(inactive)

- pEx-His-FluA-LongLinker-Fok(inactive)

- pEx-His-FluA-MiddleLinker-Fok(inactive)

- pEx-His-FluA-ShortLinker-Fok(inactive)

- pEx-His-FluA-SplitLinker-Fok(inactive)

- pEx-His-Dig

- pMA

with NgoMIV and SpeI

<ul style="margin-left: 40px;"> <li>digest of - pMA with XbaI and AgeI </li> </ul> <ul style="margin-left: 40px;"> <li>Ligation of: - Strep-Dig-LongLinker-Fok(inactive)</li> </ul> - Strep-Dig-MiddleLinker-Fok(inactive)

- trep-Dig-ShortLinker-Fok(inactive)

- Strep-Dig-SplitLinker-Fok(inactive)

- His-Dig-SplitLinker_Fok(inactive)

- His-Dig-SplitLinker_Fok(active)

- Strep-Dig-SplitLinker-Fok(active)

- His-Dig-LongLinker-Fok(inactive)

- His-FluA-LongLinker-Fok(inactive)

- His-FluA-MiddleLinker-Fok(inactive)

- His-FluA-ShortLinker-Fok(inactive)

- His-FluA-SplitLinker-Fok(inactive)

into pMA

and

- Strep-Dig-LongLinker-Fok(inactive)

- Strep-Dig-MiddleLinker-Fok(inactive)

- trep-Dig-ShortLinker-Fok(inactive)

into new pEX

<ul style="margin-left: 40px;"> <li>inoculation of: -pMASplitFokA clone1 19.08.09</li> </ul> -pMAFokA clone2 05.08.09 (new)

-pMAFoki clone2 05.08.09 (new)

-pMASplitFoki clone2 06.08.09 (new)

-pMAshortLi clone2 10.09.09

-pMAmiddelLi clone2 10.09.09

-pMAlongLi clone1 10.09.09

-pMAlongLiFokA clone1 10.09.09

-pMAlongLiFoki clone1 10.09.09

-pMAFokA clone2 05.08.09 (old)

-pMAFoki clone2 05.08.09 (old)

-pMASplitFoki clone2 06.08.09 (old)

<ul style="margin-left: 40px;"> <li>inoculation and plasmid preperation: -pEX_strepFluA </li> <li>Quenching test with HisFluASplitFoki and fluorescin-tagged oligos </li> <li>BB Ago Pcr via Taq </li> <li>Started makin new VCS M13 Phages see Protocol day 1 </li> </ul> <h3 style="margin-left: 40px;">Plan for 04.10.09

<ul style="margin-left: 40px;"> <li>inoculation of - pMA-Strep-Dig-LongLinker-Fok(inactive)</li> </ul> - pMA-Strep-Dig-MiddleLinker-Fok(inactive)

- pMA-Strep-Dig-ShortLinker-Fok(inactive)

- pMA-Strep-Dig-SplitLinker-Fok(inactive)

- pMA-His-Dig-SplitLinker_Fok(inactive)

- pMA-His-Dig-SplitLinker_Fok(inactive)

- pMA-Strep-Dig-SplitLinker-Fok(active)

- pMA-His-Dig-LongLinker-Fok(inactive)

- pMA-His-FluA-LongLinker-Fok(inactive)

- pMA-His-FluA-MiddleLinker-Fok(inactive)

- pMA-His-FluA-ShortLinker-Fok(inactive)

- pMA-His-FluA-SplitLinker-Fok(inactive)

into LB-Amp

<ul style="margin-left: 40px;"> <li>digestion of pEx-Strep-Flua with AgeI and PstI </li> <li>digestion of pMA-Short/Middle/Long/Split-Fok(active and inactive) </li> <li>ligation of the above parts and transformation into RV308 </li> <li>plasmid preparation of... -pMASplitFokA </li> </ul> -pMAFokA

-pMAFoki

-pMASplitFoki

-pMAshortLi

-pMAmiddelLi

-pMAlongLi

-pMAlongLiFokA

-pMAlongLiFoki

-pMAFokA (old)

-pMAFoki (old)

-pMASplitFoki (old)

<h3 style="margin-left: 40px;">04.10.09, Laura, Anika, Max

<ul style="margin-left: 40px;"> <li> Plasmid preparation of -pMASplitFokA </li> </ul> -pMAFokA

-pMAFoki

-pMASplitFoki

-pMAshortLiFokA

-pMAshortLiFoki

-pMAmiddelLiFokA

-pMAmiddelLiFoki

-pMAlongLiFokA

-pMAlongLiFoki

-pMAFokA (old)

-pMAFoki (old)

-pMASplitFoki (old)

clone 1 and 2,respectively - results see notebook

<ul style="margin-left: 40px;"> <li> Glycerolstock of </li> </ul> -pMASplitFokA

-pMAFokA

-pMAFoki

-pMASplitFoki

-pMAshortLiFokA

-pMAshortLiFoki

-pMAmiddelLiFokA

-pMAmiddelLiFoki

-pMAlongLiFokA

-pMAlongLiFoki

-pMAFokA (old)

-pMAFoki (old)

-pMASplitFoki (old)

clone 3

<ul style="margin-left: 40px;"> <li> Pellets of</li> </ul> -pMASplitFokA

-pMAFokA

-pMAFoki

-pMASplitFoki

-pMAshortLiFokA

-pMAshortLiFoki

-pMAmiddelLiFokA

-pMAmiddelLiFoki

-pMAlongLiFokA

-pMAlongLiFoki

-pMAFokA (old)

-pMAFoki (old)

-pMASplitFoki (old)

clone 1 and 2,respectively - stored in Pelletbox, -20&deg;C

<ul style="margin-left: 40px;"> <li>Digest of 1. pEXHisDigMiddleLFoki 2. pMaScFaantiNIP 3. prep of Ligation pEX+CAT of 09.07.09 old 4. pMAShortLFoka</li> </ul> 5. pMAShortLFoki

6. pMAMiddleLFoka

7. pMAMiddleLFoki

8. pMALongLFoka

9. pMALongLFoki

10.pEXStrepFlua

<ul style="margin-left: 40px;"> <li>Preparative Gel of Digest</li> </ul>

<ul style="margin-left: 40px;"> <li>Gelextraction</li> <li>Ligation of</li> </ul> 1.pMAHisDigMiddleFoki

2.pMACAT

3.pEXStrepFluAShortLFokA

4.pEXStrepFluAShortLFoki

5.pEXStrepFluAmiddleLFokA

6.pEXStrepFluAMiddleLFoki

7.pEXStrepFluALongLFokA

8.pEXStrepFluALongLFoki

9.JS 419StrepDigSplitFokA

10.JS 419HisDigSplitFokA

11.JS 418HisDigSplitFokA

Approach: 8&micro;l H2O, 3 &micro;l Vector, 6&micro;l Insert, 2&micro;l Quick Ligase Buffer, 1 &micro;l Quick Ligase, 15 min, Room Temp.

<ul style="margin-left: 40px;"> <li>Dephosphorylation of pBAD Vector: </li> </ul> Approach: 2&micro;l Eluat, 3,1 &micro;l Fast Ap Buffer 10x, 1&micro;l Fast Ap, 10 min. 37&deg;C, 5 min. 75&deg;C

<ul style="margin-left: 40px;"> <li>Transformation of</li> </ul> 1.pMAHisDigMiddleFoki (RV)

2.pMACAT(RV)

3.pEXStrepFluAShortLFokA(RV)

4.pEXStrepFluAShortLFoki(RV)

5.pEXStrepFluAmiddleLFokA(RV)

6.pEXStrepFluAMiddleLFoki(RV)

7.pEXStrepFluALongLFokA(RV)

8.pEXStrepFluALongLFoki(RV)

9.pBAD (RV)

10.pJS 419StrepDigSplitFokA(XBL)

11.pJS 419HisDigSplitFokA(XBL)

12.pJS 418HisDigSplitFokA(XBL)

<ul style="margin-left: 40px;"> <li>test digest of plasmid preps from today -&gt; run on a gel with digest pET39, xbaI, ecoRI from Tobi </li> <li>starter culture of pEx_DsbA_HisDigSplitFoka -&gt; modified expression has to be done tomorrow </li> <li>inoculation of pJS418 and pJS419 for glycerolstocks tomorrow </li> <li>Taq AGO BB PCR did not work </li> <li>Phageproduction day 2 </li> </ul> <h3 style="margin-left: 40px;">05.10.09, Laura, Manu, Julia, Christophe, Caro, Hannes, Gerrit, Anika

<ul style="margin-left: 40px;"> <li> 3l LB and 2L LB Agar </li> <li>Periplasma Project </li> <li>digest pExHisFluASplitFoki (prep from 24.08.09) and RBS_StrepDigSplitFoka (prep from 29.09.09) </li> <li>test digests of pMAFoka, pMALongliFoki, pMAFoka old, pMAFoki old, pExHisFluASplitFokiStrepDigSplitFoka </li> <li>1) Gelextraction</li> </ul> --&gt; Gelbild

1) Vector: pEXHisFluSplitFoki

2)Insert: RBSStrepDigSplitFokA

3)PET39b+

<ul style="margin-left: 40px;"> <li>2)Ligation</li> </ul> -10ul 2 fold buffer

- 6 &micro;l Insert

-3&micro;l Vector

-1&micro;l Quick Ligase

--&gt; 15 min at RT

Vector:pEXHisFluSplitFoki + Insert:RBSStrepDigSplitFokA

<ul style="margin-left: 40px;"> <li>3)Transformation</li> </ul> -5 &micro;l DNA of Ligation + BL21d3 and XBL

<ul style="margin-left: 40px;"> <li>Mini prep with with Qiagen Spin Miniprep Kit of - pMA-Strep-Dig-LongLinker-Fok(inactive)1,2,3</li> </ul> - pMA-Strep-Dig-MiddleLinker-Fok(inactive)1,2,3

- pMA-Strep-Dig-ShortLinker-Fok(inactive)1,2,3

- pMA-Strep-Dig-SplitLinker-Fok(inactive)1,2,3

- pMA-His-Dig-SplitLinker_Fok(inactive)1,2,3

- pMA-His-Dig-SplitLinker_Fok(inactive)1,2,3

- pMA-Strep-Dig-SplitLinker-Fok(active)1,2,3

- pMA-His-Dig-LongLinker-Fok(inactive)1,2,3

- pMA-His-FluA-LongLinker-Fok(inactive)1,2,3

- pMA-His-FluA-MiddleLinker-Fok(inactive)1,2,3

- pMA-His-FluA-ShortLinker-Fok(inactive)1,2,3

- pMA-His-FluA-SplitLinker-Fok(inactive)1,2,3

- pMA-Dbsa 1,2,3

<ul style="margin-left: 40px;"> <li>Made Glycerolstocks of </li> <li>700&micro;l Cellsuspension+ 300&micro;l Glycerol - pMA-Strep-Dig-LongLinker-Fok(inactive)1,2,3</li> </ul> - pMA-Strep-Dig-MiddleLinker-Fok(inactive)1,2,3

- pMA-Strep-Dig-ShortLinker-Fok(inactive)1,2,3

- pMA-Strep-Dig-SplitLinker-Fok(inactive)1,2,3

- pMA-His-Dig-SplitLinker_Fok(inactive)1,2,3

- pMA-His-Dig-SplitLinker_Fok(active)1,2,3

- pMA-Strep-Dig-SplitLinker-Fok(active)1,2,3

- pMA-His-Dig-LongLinker-Fok(inactive)1,2,3

- pMA-His-FluA-LongLinker-Fok(inactive)1,2,3

- pMA-His-FluA-MiddleLinker-Fok(inactive)1,2,3

- pMA-His-FluA-ShortLinker-Fok(inactive)1,2,3

- pMA-His-FluA-SplitLinker-Fok(inactive)1,2,3

- pMA-Dbsa 1,2,3

<ul style="margin-left: 40px;"> <li>Test Digestion of </li> <li>put in each sample: 5&micro;l DNA, 2,5&micro;l H2O, 1&micro;l Buffer 3, 1&micro;l EcoRI, 1&micro;l PstI, 1&micro;l BSA - pMA-Strep-Dig-LongLinker-Fok(inactive)3</li> </ul> - pMA-Strep-Dig-MiddleLinker-Fok(inactive)1

- pMA-Strep-Dig-ShortLinker-Fok(inactive)1

- pMA-Strep-Dig-SplitLinker-Fok(inactive)3

- pMA-His-Dig-SplitLinker_Fok(inactive)1

- pMA-His-Dig-SplitLinker_Fok(active)2

- pMA-Strep-Dig-SplitLinker-Fok(active)1

- pMA-His-Dig-LongLinker-Fok(inactive)1

- pMA-His-FluA-LongLinker-Fok(inactive)3

- pMA-His-FluA-MiddleLinker-Fok(inactive)3

- pMA-His-FluA-ShortLinker-Fok(inactive)1

- pMA-His-FluA-SplitLinker-Fok(inactive)2

- pMA-Dbsa 2

<ul style="margin-left: 40px;"> <li> Transformation of</li> </ul> - XBL pEXHisFluASplitFokIRBSStrepDigSplitFokA

- BL21de3 pEXHisFluASplitFokIRBSStrepDigSplitFokA

<ul style="margin-left: 40px;"> <li>Expressionsculture of pEx_DsbA_HisDigSplitFoka in 6X2l flasks in 600ml DYT medium each - induced with 0,7mM IPTG and took samples T0-T5</li> </ul> - centrifuged in buckets 17', 4000rpm, 4&deg;C

- eluted in 20 ml TES in each bucket

....

<ul style="margin-left: 40px;"> <li>PET 39 b+ ssDNA "PCR" with new template from digestion of yesterday -&gt; very few product, as well as non specific ones </li> <li>PET 39 b+ ssDNA "PCR" with more cycles and higher annealing temperature </li> </ul> <h3 style="margin-left: 40px;">06.10.09, Laura, Manu, Julia, Christophe, Caro, Hannes, Gerrit, Anika

<ul style="margin-left: 40px;"> <li>digest - pBAD vector </li> </ul> DNA: 16.8&micro;l

enzyme: 1&micro;l AgeI

buffer: 2&micro;l buffer 1

- pBAD insert

DNA: 16.8&micro;l

enzyme: 1&micro;l XmaI

buffer: 2&micro;l buffer 4

BSA (10X): 2&micro;l

- pJS 418/419

DNA: 10&micro;l

enzyme: 1&micro;l PstI and 1.5 &micro;l Xba/

buffer: 3&micro;l buffer 3

BSA (10X): 3&micro;l

- pExStrepDigSplitFoka/pExHisDigSplitFoka

DNA: 16.8&micro;l

enzyme: 1&micro;l PstI and 1.5 &micro;l Xba/

buffer: 3&micro;l buffer 3

BSA (10X): 3&micro;l

<ul style="margin-left: 40px;"> <li>testdigest of pExHisFluASplitFoki_StrepDigSplitFoka DNA: 5&micro;l</li> </ul> enzyme: 1&micro;l PstI and 1.5 &micro;l Xba/

buffer: 1&micro;l buffer 3

BSA (10X): 1&micro;l

<ul style="margin-left: 40px;"> <li>preparative gels of digests </li> </ul>

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

<ul style="margin-left: 40px;"> <li>testdigest of pMAdsba DNA: 5&micro;l</li> </ul> enzyme: 1&micro;l PstI and 1.5 &micro;l Xba/

buffer: 1&micro;l buffer 3

BSA (10X): 1&micro;l

<ul style="margin-left: 40px;"> <li>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 0.45&micro;m and 0.22&micro;m filter </li> </ul> <ul style="margin-left: 40px;"> <li>Ligation - Dephphorylation of pBAD Gelex</li> </ul> - 1&micro;l Fast AP

- 5.5&micro;l fast AP buffer

-1.5&micro;l water

--&gt; Solution was given to eluat - for each ligation:

- 6&micro;l Insert

-3&micro;l Vetor

-1&micro;l Quickligase

-10&micro;l buffer

--&gt; pBAD

<ul style="margin-left: 40px;"> <li> Insert (Dummy)</li> </ul> --&gt; pJS419+HisDigSplitFoka --&gt;pJ418+HisDigSplitFoka

<ul style="margin-left: 40px;"> <li> Transformation --&gt; pBAD </li> <li> Insert (Dummy)</li> </ul> --&gt; pJS419+HisDigSplitFoka --&gt;pJ418+HisDigSplitFoka - in XLblue

-pBAD was plated on AMP plates

- Both pJS... were plated on CM plates

<ul style="margin-left: 40px;"> <li> Ligation and Transformation of </li> </ul> Ligation:per sample 8&micro;l H2O; 3&micro;l vector; 6&micro;l insert; 2&micro;l Quick Ligase buffer; 1&micro;l Quick Ligase Transformation:Defrost competent cells on ice:(100 &micro;l); add of the ligation: 5 &micro;l; DNA and cells: mix softly by knocking; <b>Incubation on ice for:</b> 20-30 min; Heat shock at: 42&deg;C for 40 sec; Cool off on ice for: 5 min; add sterile LB(or dyt)medium: 900 &micro;l; <b>Incubation in(shaker)at:</b> 37&deg;C for 60-70 min; <b>Plate cells on LB+antibiotic plates:</b> ampicillin; ligation: 2 plates: 1. 50&micro;l cells <b>2. centrifuge @ 2000 rpm 3 min, discard supernatant, resuspend the restcells and plate out:</b> -pMA HisDig Middle Linker Foki

-pMA CAT

-pEX Strep FluA SL FokA

-pEX Strep FluA LL Foki

-pEX Strep FluA ML Foki

-pEX Strep FluA ML Foka

-pEX Strep FluA LL Foka

<ul style="margin-left: 40px;"> <li>Sequencing: 27&micro;l H2O; 3&micro;l DNA -pMA Dbsa clone 2</li> </ul> -pMA HisDigSplitFokA clone 2

-pMA HisFluASL Foki clone 1

-pMA HisFluASplitFoki clone 2

-pMA StrepDigLLFoki clone 3

-pMA HisFluaMLFoki clone 3

-pMA StrepDigSplitFokA clone 1

-pMA StrepDigMLFoki clone 1

-pMA StrepDigSLFoki clone 2

-pMA HisDigLLFoki clone 1

-pMA StrepDigSplitFoki clone 3

-pMA HisDigSplitFoki clone 1

-pMA HisFluaLLFoki clone 3

<ul style="margin-left: 40px;"> <li>Inoculation of ER2738 M13: 250ml Erlenmeyerkolben+ 50ml LB+TET in 37&deg;c room overnight</li> <li>Yesterdays improved pcr resulted in a lot of product, but still unspecific ones.. made new one with even higher annealing temperature (69&deg;C) and a bit less cycles (35) </li> <li>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 </li> <li>Started dialysis to transfer the leftover AGO-proteins into the assay buffer </li> </ul> FokI wildtype cutting

Hybridize M13 ssDNA with Fok control oligos 2 and 3 The hybridization was also done with just Fok control 2 and with no oligos at all. Incubation with the thermocycler and the program ORIGAMI0 (heat to 95°C and cool down slowly to 37°C over 1 hour).

10 µl of the hybridized DNA were used for a cutting experiment with 1,2 µl buffer 4 (NEB) and 0.5 µl FokI (wildtype Fok from NEB). The digest was incubated for 30 minutes at 37°C.

Lane 1: GeneRuler DNA Ladder Mix (Fermentas, 6 µl); Lane 2: Untreated M13 ssDNA (250 ng); Lane 3: Digest of M13 ssDNA with FokI and without oligo; Lane 4: Digest of M13 ssDNA with FokI and with one oligo; Lane 5: Digest of M13 ssDNA with FokI and two oligos.

<h3 style="margin-left: 40px;">07.10.09 Laura,Christoph, Hannes, Timo, Julia, Caro, Anika

<ul style="margin-left: 40px;"> <li>SDS gel of protein purification of HisDigSplitFoka (periplasm) from 06.10.09 </li> </ul> <ul style="margin-left: 40px;"> <li> Inoculation 4 Clones respectively:</li> </ul> - 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 10&micro;l

-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

<ul style="margin-left: 40px;"> <li> Starter culture of pEx-DsbAHisDigSplitFoka in Bl21de3 </li> <li>two step PCR assembly of DsbA, His_Fos, and SplitFoka - program name: Assembly</li> </ul> - three different samples: 1. without DMSO, 2. with DMSO, 3. without DMSO and with last primers just added after first step

<ul style="margin-left: 40px;"> <li>preparative gel of the PCR samples -&gt; primer haven't been diluted an probably made all secondary structures, has to be repeated </li> <li>digest of pBAD with AgeI and new PCR with insert digested with XmaI </li> <li>preparative gel with digest and </li> <li>test digest of pJS419_HisDigSplitFoka and pJS419_StrepDigSplitFoka DNA: 5&micro;l</li> </ul> Enzymes: 0.5&micro;l of BamHI and MfeI each

Buffer: 1&micro;l buffer 4

BSA (10fold): 1&micro;l

Water: 2&micro;l

-&gt; pJS419_HisDigSplitFoka showed bands of the right size and was sequenced with primer sf_lac P1

<ul style="margin-left: 40px;"> <li>plasmidpreparation of - pEXHisFluASplitFoki</li> </ul> - pExStrepDigSplitFoka

-&gt; low concentrations

<ul style="margin-left: 40px;"> <li>digest of pExHisFluASplitFoki, pExStrepDigSplitFoka and RBS_StrepDigSplitFoka pExHisFluASplitFoki: 15&micro;l</li> </ul> Enzymes: 1&micro;l of PstI and 1.5&micro;l SpeI

Buffer: 3&micro;l buffer 2

BSA (100fold): 0.5&micro;l

Water: 9&micro;l

pExStrepDigSplitFoka: 15&micro;l

Enzymes: 1&micro;l of XbaI and 1.5&micro;l PstI

Buffer: 3&micro;l buffer 3

BSA (100fold): 0.5&micro;l

Water: 9&micro;l

pExStrepDigSplitFoka: 10&micro;l

Enzymes: 1&micro;l of XbaI and 1.5&micro;l PstI

Buffer: 3&micro;l buffer 3

BSA (100fold): 0.5&micro;l

Water: 14&micro;l

<ul style="margin-left: 40px;"> <li> Gelextraction of digest 1) Insert: pEX-Strep-Dig-Split-Foka( digested with xba and pst) </li> </ul> 2)Vector: pEX-His-Flua-Split-Foki (digested with spe, pst)

3)PCR Product: RBS-Strep-Dig-Split-FokA(digested with pst, xba)

<ul style="margin-left: 40px;"> <li>preparative gels of digests </li> </ul>

RBS PCR Product: m= 90 mg

vector: 0 0 130 mg

<ul style="margin-left: 40px;"> <li> Ligation - 6&micro;l Insert</li> </ul> -3&micro;l Vetor

-1&micro;l Quickligase

-10&micro;l buffer

pEX-Strep-Dig-Split-Foka-RBS-Strep-Dig-Split-Foka

<ul style="margin-left: 40px;"> <li>Transformation - All in XL1blue</li> </ul> - Vector: pEX-strep-Duig-Split-Foka

- Ligation: pEX-Strep-Dig-Split-Foka-RBS-Strep-Dig-Split-Foka

<ul style="margin-left: 40px;"> <li>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 </li> <li>prepaired ELISA with anti-flag antibodies </li> <li>made electro competent cells for transformation with the ago phagmidbibliothek </li> </ul> <h3 style="margin-left: 40px;">08.10.09 Manu, Christoph, Julia, Caro, Timo, Hannes

<ul style="margin-left: 40px;"> <li>Poured SDS-Gels</li> <li>Phage ss DNA -Over night cultured ER2738 in 100ml LB+Tet dilute on OD600=0,4 in 50ml LB+Tet</li> </ul> -Transfomation with 3&micro;l M13 Phage stock (-80&deg;) at 10:30Uhr, incubated for 4-5 hours at 37&deg;C in shaker

-Centrifuge in 50ml Falcon tube at 5000rpm for 20 min, 4&deg;C

-Phages are in the supernatant, add 1/7 PEG/NaCl (for 50ml culture 7ml precipitation over night on ice in -4&deg;C room

<ul style="margin-left: 40px;"> <li>Miniprep - 2 clones respectively:</li> </ul> -A = pEX strep dig split foka

<ul style="margin-left: 40px;"> <li>Finished Phage production(see protocol day 2): We obtained approximately 3.8</li> <li>10^12 pJS_TorA-flag-AGO-noAmber-CDg3p (449) and 1.1</li> <li>10^13 pJS_TorA-flag-AGO-CDg3p (448) -&gt; aplyed all of them to the anti-Flag ELISA</li> </ul>

<ul style="margin-left: 40px;"> <li>Anti-Flag ELISA was successfull with a slight but detctable signal via anti M13 VCS Antibodies (with peroxidase):</li> </ul> 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.

<ul style="margin-left: 40px;"> <li> Made new PCR for ssDNA from pET39b+ fragment and gained approximately 200 ng of ssDNA after PCR and gelextraction </li> <li>digest of pExHisFluASplitFoki, pExStrepDigSplitFoka and RBS_StrepDigSplitFoka pExHisFluASplitFoki: 15&micro;l</li> </ul> Enzymes: 1&micro;l of PstI and 1.5&micro;l SpeI

Buffer: 3&micro;l buffer 2

BSA (100fold): 0.5&micro;l

Water: 9&micro;l

pExHisDigSplitFoka: 15&micro;l

Enzymes: 1&micro;l of XbaI and 1.5&micro;l PstI

Buffer: 3&micro;l buffer 3

BSA (100fold): 0.5&micro;l

Water: 14&micro;l

RBSStrepDigSplitFoka: 5&micro;l

Enzymes: 1&micro;l of XbaI and 1.5&micro;l PstI

Buffer: 3&micro;l buffer 3

BSA (100fold): 0.5&micro;l

Water: 19&micro;l

-&gt;made two digest of PCR construct

<ul style="margin-left: 40px;"> <li> Gelextraction of digest 1) Insert: His-Dig-Split-Foka( digested with xba and pst) </li> </ul> 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

<ul style="margin-left: 40px;"> <li>preparative gels of digests -&gt;see picture</li> </ul>

<ul style="margin-left: 40px;"> <li>ligation of -pEX-His-Flua-Split-Foki_His-Dig-Split-Foka</li> </ul> -pEX-His-Flua-Split-Foki_RBS_Strep-Dig-Split-Foka (from gelex)

-pEX-His-Flua-Split-Foki_RBS_Strep-Dig-Split-Foka (from PCR purification)

<ul style="margin-left: 40px;"> <li>transformation of ligations </li> <li>inoculation of pJS419_HisDigSplitFoka in LB + chloramphenicol </li> <li>new two step PCR with all three plasmids for Fos construct (pMADsbA, pMAFos, pMASplitFoka, this time with right primer dilution and preparative agarose gel -&gt;interpretation: no band of 921bp was visible</li> </ul>

<ul style="margin-left: 40px;"> <li>one step PCR of pMADsbA, pMAFos, pMASplitFoka separated and preparative agarose gel -&gt;see picture</li> </ul> -&gt;interpretation: pMAFos and pMASplitFoka showed bands of the right size (230bp and 657bp respectively), of pMADsbA no product was visible

<ul style="margin-left: 40px;"> <li>new one step PCR of pMADsba with newly prepared 1:1000 dilution </li> <li>test digest of plasmidpreparations from today -&gt;see picture</li> </ul> <h3 style="margin-left: 40px;">09.10.09 Manu, Julia, Caro, Laura, Christopherus, Hannes, Timo, Max, Anika

<ul style="margin-left: 40px;"> <li>Phage ss DNA</li> </ul> -Over night in 4&deg;C room precipitated phages: centrifuged for 20 min at 5000rpm and 4&deg;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 170&micro;l PEG/NaCl and leave for 1 hour on ice

<ul style="margin-left: 40px;"> <li>Miniprep - pJS 419-his-dig-split-foka (clon 1+2)</li> </ul> - Glycerolstock of clon1 and 2

- 300&micro;l Glycerol

- 700 &micro;l culture

- Pellets from clones 3,5,6

Nanodrop data:

pJS 419-his-dig-split-foka-clon1= 509.5 ng/&micro;l

pJS 419-his-dig-split-foka-clon2 =468 ng/&micro;l

<ul style="margin-left: 40px;"> <li>Inoculation of ER2738 in 2x 2L conical flask with 1L LB+1ml Tet, over night in 37&deg;C room </li> <li>send to sequencing:</li> </ul> pMA Dig Plasmidprep 22.07.09 Timo1

pEx StrepDigSplitFoka Plasmidprep clone1 08.10.09 Timo2

pEx HisFluaSplitFoki Plasmidprep clone1 08.10.09 Timo3

pEx StrepFluAMiddleLinkerFoki Plasmidprep clone1 08.10.09 Timo4

pEx StrepFluAMiddleLinkerFoka Plasmidprep clone1 08.10.09 Timo5

pEx StrepFluALongLinkerFoki Plasmidprep clone1 08.10.09 Timo6

pMA Cat plasmidprep clone1 08.10.09 Timo7

pJs 419 HisDigSplitFoka plasmidprep09.10.09 clone1 Timo8

pJs 419 HisDigSplitFoka plasmidprep09.10.09 clone2 Timo9

pJs 419 HisDigSplitFoka plasmidprep09.10.09 clone5 Timo12

<ul style="margin-left: 40px;"> <li> concentrated pET39b+ ssDNA via sodiumacetat and ethanol precipitation </li> <li> electrical trafo of the 449 ago phagmid bibliothek into XL1 </li> <li> prepaired immutubes with streptavidin for ago phages test panning tomorrow </li> </ul> <h3 style="margin-left: 40px;">10.10.09 Julia, Caro, Laura, Christoph

<ul style="margin-left: 40px;"> <li>Measured OD600 of over night ER2738 culture abs.1)0.218 (1:10);2)0.23 (1:10), diluted to OD600 abs.0,1 in 1000ml LB+Tet</li> <li>Measured OD600 of Christophs culture:abs. 0.37 (1:10), diluted to abs.0.07 in 60ml DYT+Tet+CM </li> <li>Growed Er2738 up to OD600 abs.0.6 8x 250ml in 1L Erlenmeyer flasks and infected with 15&micro;l M13 phage, shake for 4 hours at 37&deg;C </li> <li>Decanted to 16x50ml falcon tubes, centrifuged for 20min at 5000rpm and 4&deg;C, decanted supernatant into new 16x50ml falcons with each 7ml PEG/NaCl </li> <li>Precipitaion over night in 4&deg;C room </li> <li>plasmidpreparation of - pBADQuick clones 1-6</li> </ul> - pBADT4 clones 1-6

- pMAYFP clone 1-2

- pMACFP clone 1

- pMASplitlinker clones 1-2

- HisTag clones 1-2 (but test digest was negative -&gt; thrown away)

<ul style="margin-left: 40px;"> <li>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: 10&micro;l</li> </ul> Enzymes: 1&micro;l of NgoMIVI and 1.5&micro;l PstI

Buffer: 3&micro;l buffer 1

BSA (10fold): 3&micro;l

Water: 10,5&micro;l

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: 10&micro;l each

Enzymes: 1&micro;l of AgeI and 1.5&micro;l PstI

Buffer: 3&micro;l buffer 1

BSA (10fold): 3&micro;l

Water: 10,5&micro;l

<ul style="margin-left: 40px;"> <li>PCR assembly with pMADsbA, different approaches (Taq Polymerase or Phusion Polymerase, differnt Tm) and combination PCR with pMADsba and pMAFos together </li> <li>analytical gel of PCRs -&gt; didn't work </li> <li>preparative gels of digests -&gt; see picture -&gt; no inserts with pExDSba_Foka constructs because they have no NgoMIV site any more </li> <li>ligation of -pMAStrepDigShortLiFoka</li> </ul> -pMAStrepDigMiddleLiFoka

-pMAStrepDigLongLiFoka

-pExStrepDigShortLiFoka

-pExStrepDigMiddleLiFoka

-pExStrepDigLongLiFoka

-pMAHisDigShortLiFoka

-pMAHisDigMiddleLiFoka

-pMAHisDigLongLiFoka

-pMACATNd4 (MQI)

-pMACATNd4 (MQII)

-pMA Kontrolle (M)

-&gt; in XLBlue

- pEx_CATNd4 (EQI)

- pEx_CATNd4 (EQII)

- pEx_Kontrolle(E)

-&gt;RV308 Insert: 6&micro;l

vector: 3&micro;l

Ouick ligase buffer: 10&micro;l

Quick ligase: 1&micro;l

<ul style="margin-left: 40px;"> <li>transformation of ligations in XBL on LB/Amp/1%glucose plates </li> <li>cotransformation of </li> <li>testdigest of the pET39b+ ssDNA. looks like a success... see 4pk in picture</li> </ul>

<ul style="margin-left: 40px;"> <li>cotransformation of pExHisFluASplitFoki and pJSStrepDigSplitFoka in XBL on LB/Amp/CM/1%glucose plates </li> <li>inoculation of pExDsbAStrepDigSplitFoka and pExDsbAHisDigSplitFoka, glystock from 02.10.09 </li> </ul> <h3 style="margin-left: 40px;"> 11.10.09, Timo, Hannes, Max, Anika

<ul style="margin-left: 40px;"> <li> Digestion of </li> </ul> 1.pEXHisFluA (27.09.09, Klon1), XbaI - PstI

2.pMAFos_HlsbZip, XbaI - AgI

3.pMASplitFoka (04.10.09, Klon2), NgoIV - PstI

4.pEXDsbaHisDigSplitFoka (02.10.09, Klon1), XbaI - PstI

5.pEXDsbaStrepDigSplitFoka (02.10.09, Klon1), XbaI - PstI

6.pMA_BB057, XbaI - PstI (01.10.09)

<ul style="margin-left: 40px;"> <li>gel extraction of - pMA</li> </ul> - pEx

- ...

<ul style="margin-left: 40px;"> <li> Testdigestion of </li> </ul> 7.pMA_YFP 2 (10.10.09, Caro)

8.pMA_CFP (10.10.09, Caro)

9.pMASplitLi1 (10.10.09, Caro)

10.pMASplitLi2 (10.10.09, Caro)

...image..

<ul style="margin-left: 40px;"> <li> 1% Agarosegel of constructs above </li> <li> Phage breeding day 2 </li> <li> Starter culture of pEx_DsbA_StrepDigSplitFoka (Bl21de3) </li> <li> plasmid prep of pEx_DsbA_StrepDigSplitFoka </li> <li> plasmid prep of pEx_HisDigSplitFoka </li> <li> 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 </li> </ul> <h3 style="margin-left: 40px;"> 12.10.09, Laura, Caro, Christoph, Anika, Hannes, Julia, Timo, Gerrit

<ul style="margin-left: 40px;"> <li> protein expression of pEx_DsbA_StrepDigSplitFoka (periplasm) in BL21de3 at 22&deg;C. </li> <li> made 5 litres of DYT </li> <li>digest of pMASplitFoka clone 1 and 2 from 04.10.09 DNA:10&micro;l Enzymes: 1&micro;l of NgoMIVI and 1.5&micro;l PstI</li> </ul> Buffer: 3&micro;l buffer 1

BSA (100fold): 0.5&micro;l

Water: 14.5&micro;l

<ul style="margin-left: 40px;"> <li> Plasmidprep. of: </li> </ul> 1.pMAFokA (old)

2.pMA-CAT Ndelta4 (MQII)1

3.pMA-CAT Ndelta4 (MQII)2

4.pMA-CAT Ndelta4 (MQII)3

5.pMA-CAT Ndelta4 (MQII)4

6.pMA-CAT Ndelta4 (MQII)5

7.pMA-CAT Ndelta4 (MQII)6

8.pMAFoka clone 1 from 04.10.

<ul style="margin-left: 40px;"> <li>Made new BB-AGO PCR using digested AGO Gene without the vector-&gt; still no expected bands to be seen... </li> <li>Made new PCR to generate more pET39b+ ssDNA because yesterdays had insufficient concentration for the AGO cleavage assay </li> <li>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 </li> <li>Send to sequencing:Protocols</li> </ul> Julia 1-6:

1)pBADQuick clone 3

2)pBAD T4 clone 2

3)pBAD T4 clone 1

4)pMA YFP clone 1

5)pMA CFP clone 1

6)pMA Split Linker clone 2

Gerrit1: pMA_cat-Nd4

<ul style="margin-left: 40px;"> <li>Prepared ELISAProtocols and two immuno tubes for Panning Simulation for tomorrow </li> <li>made chemical competent cells of cotransformed XBlue with pJS419HisDigSplitFoka, pExHisFluASplitFoki -&gt;will prepare electrocompetent cells tomorrow</li> <li>inoculation of cotransformed XBlue with pJS419HisDigSplitFoka, pExHisFluASplitFoki in LB+Amp+Tet+CM and pMAFos in LB+Amp </li> <li>PCR of pMADsba repeated with different (higher) template dilutions -&gt;analytical gel (see picture)</li> </ul> -&gt;didn't work again

<ul style="margin-left: 40px;"> <li>poured IPTG/XGal plates for in vivo plaque assay test run </li> <li>transformation of:</li> </ul> - pBAD_FokA (Quick)

- pBAD_CAT(Quick)

- pBAD_FokA (T4)

- pBAD_CAT (T4)

- pEX_FokA+YFP

- pEX_DsbA+FokA+YFP

into BL21 de3 gold

Plates: LB+ AMP +1%Glucose

<ul style="margin-left: 40px;"> <li>cultivation of pma strep dig split foka</li> </ul> pma long linker

pma his flua split foki

<h3 style="margin-left: 40px;"> 13.10.09, Laura, Caro, Christoph,Manu, Hannes, Julia

<ul style="margin-left: 40px;"> <li>Miniprep -pMA-fos 1</li> </ul> -pMA-fos 2

-pMA his flua split fokI

- pMA strep dig split foka

-pMA long linker

<ul style="margin-left: 40px;"> <li>testdigest of Plasmidprep from today 15 &micro;l for 6&micro;l loading dye -buffer 2 1&micro;l</li> </ul> -xbaI 0.5&micro;l

-pstI 0.75&micro;l

-DNA 2 &micro;l

-water 9.75&micro;l

-BSA 1&micro;l

--&gt; Gelbild - Glycerolstocks are in in box of 5/10/2009

<ul style="margin-left: 40px;"> <li>made electrocompetent XL1 Blue cotransformed cells (pExHisFluASplitFoki and pJS419HisDigSplitFoka) -&gt;stored in -80&deg;C </li> <li>culture of ER2738 in LB Tet for cotransformation in afternoon </li> <li>test in vivo assay - electroporation with new electrocompetent cells and M13 ssDNA at 1.7kV</li> </ul> - 1.5h on 37&deg;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 -&gt; 37&deg; shaker

<ul style="margin-left: 40px;"> <li>inoculation of -pMASplitFoka clone 1 from prep 04.10.09</li> </ul> -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

<ul style="margin-left: 40px;"> <li>protein purification of DsbA_StrepDigSplitFoka with Strep column </li> <li>digestion, 1% agarose gel and gel extraction of - pMA-LongLinker-FokA (10.09., clone 1) with EcoRI + SpeI</li> </ul> - pMA-His-FluA-Split-FokI (13.10.) with SpeI + PstI

- pMA-SplitLinker-FokA (04.10., clone 2) with NgoMIV + PstI =&gt; 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/&micro;l) with XbaI + AgeI - pMA (01.10., c=500ng/&micro;l) with EcoRI + SpeI

<ul style="margin-left: 40px;"> <li>ligations: Vector: 3 &micro;l pMA (01.10., c=500ng/&micro;l) with EcoRI + SpeI</li> </ul> Insert: 6 &micro;l LongLinker-FokA (10.09., clone 1) with EcoRI + SpeI

Buffer: 10 &micro;l, QuickLigase Buffer NEB

QuickLigase: 1 &micro;l

Vector: 3 &micro;l pMA-His-Dig (24.08., clone 2) with AgeI + PstI

Insert: 6 &micro;l ShortLinker-FokA (04.10., clone 2) with NgoMIV + PstI

Buffer: 10 &micro;l, QuickLigase Buffer NEB

QuickLigase: 1 &micro;l

Vector: 3 &micro;l pMA-His-Dig (24.08., clone 2) with AgeI + PstI

Insert: 6 &micro;l MiddleLinker-FokA (04.10., clone 1) with NgoMIV + PstI

Buffer: 10 &micro;l, QuickLigase Buffer NEB

QuickLigase: 1 &micro;l

Vector: 3 &micro;l pMA-Strep-Dig (25.09., clone 2) with AgeI + PstI

Insert: 6 &micro;l ShortLinker-FokA (04.10., clone 2) with NgoMIV + PstI

Buffer: 10 &micro;l, QuickLigase Buffer NEB

QuickLigase: 1 &micro;l

Vector: 3 &micro;l pMA-Strep-Dig (25.09., clone 2) with AgeI + PstI

Insert: 6 &micro;l MiddleLinker-FokA (04.10., clone 1) with NgoMIV + PstI

Buffer: 10 &micro;l, QuickLigase Buffer NEB

QuickLigase: 1 &micro;l

Vector: 3 &micro;l pMA (01.10., c=500ng/&micro;l) with XbaI + AgeI

Insert: 6 &micro;l Strep-FluA-MiddleLinker-FokI (08.10., clone 1) with XbaI + AgeI

Buffer: 10 &micro;l, QuickLigase Buffer NEB

QuickLigase: 1 &micro;l

Vector: 3 &micro;l pMA (01.10., c=500ng/&micro;l) with XbaI + AgeI

Insert: 6 &micro;l Strep-FluA-MiddleLinker-FokA (08.10., clone 1) with XbaI + AgeI

Buffer: 10 &micro;l, QuickLigase Buffer NEB

QuickLigase: 1 &micro;l

Vector: 3 &micro;l pMA (01.10., c=500ng/&micro;l) with XbaI + AgeI

Insert: 6 &micro;l Strep-FluA-LongLinker-FokI (08.10., Klon 1) with XbaI + AgeI

Buffer: 10 &micro;l, QuickLigase Buffer NEB

QuickLigase: 1 &micro;l

Vector: 3 &micro;l pMA (01.10., c=500ng/&micro;l) with XbaI + AgeI

Insert: 2 &micro;l ShortLinker, hybridised, with XbaI + AgeI

Buffer: 14 &micro;l, QuickLigase Buffer NEB

QuickLigase: 1 &micro;l

Vector: 3 &micro;l pMA (01.10., c=500ng/&micro;l) with XbaI + AgeI

Insert: 2 &micro;l MiddleLinker, hybridised, with XbaI + AgeI

Buffer: 14 &micro;l, QuickLigase Buffer NEB

QuickLigase: 1 &micro;l

Vector: 3 &micro;l pMA (01.10., c=500ng/&micro;l) with XbaI + AgeI

Insert: 2 &micro;l LongLinker, hybridised, with XbaI + AgeI

Buffer: 14 &micro;l, QuickLigase Buffer NEB

QuickLigase: 1 &micro;l

<ul style="margin-left: 40px;"> <li>did transformations of the ligations in RV308, plated on LB-Amp + 1% Gluc </li> <li>ELISA -Blocking</li> </ul> Washing with TBST-EDTA 5x

Loaded immunotubes (1.5ml) and wells 100&micro;l each with 0.5mM biotin-target DNA

Incubation at 20&deg;C in shaking

Incubatet Phages with Oligo for 30min at 55&deg;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&deg;C

Loaded immunotubes (each 1.5 ml) with 50&micro;l P1, 25&micro;l 449Phage+ Oligo in TBST-EDTA

Incubation in shaker at 20&deg;C

Washing of wells and immunotubes with TBST-EDTA 5x -Loaded 100&micro;l/well anti-M13 ab(1:1000), incubated for 1 hour at 20&deg;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

<ul style="margin-left: 40px;"> <li>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-&gt; 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... </li> <li>AGO assay using different targets-&gt; were not able to reproduce the presumed cutting event from Saturdays assay probably due to the lower DNA concentration </li> <li>Made ELISA with strep-biotinylated target oligo coated surface-&gt; no binding of AGO bearing phages was detected using anti-M13 HRP and ABTS </li> <li>Made test panning just like the ELISA above and eludet with TBS-MnCl2 (5mM), then TBST-MnCl2 (5mM) and then with DNase I </li> <li>Ligated product of the errore-prone PCR of the AGO-G3P construct into an phagmidvektor with the Tor-A signalling sequence</li> </ul>

<h3 style="margin-left: 40px;"> 14.10.09, Laura, Caro, Christoph, Hannes, Julia, Anika <ul> <li> Made LB-medium, 0,7% LB-Agar, 50% Glucose </li> </ul> --&gt; no differences between induced and control cells <ul> <li> SDS gel of DsbA_StrepDigSplitFoka purification from 13.10.09</li> </ul> <ul> <li>Plasmidpreparation </li> </ul> 1 = pEX-fos-split-fokA-K1 2 = pEX-fos-split-fokA-K2 3 = pMA-DsbA-strep-dig-split-fokA-K1 4 = pMA-DsbA-strep-dig-split-fokA-K2 5 = pMA-DsbA-his-dig-split-fokA-K1 6 = pMA-DsbA-his-dig-split-fokA-K2 7 = pMA-fos-split-fokA-K1 8 = pMA-fos-split-fokA-K2 9 = pBAD-fokA-K1 10= pBAD-fokA-K2 11 = pBAD-CAT-K1 12= pBAD-CAT-K2 13= pMA-shortlinker-fokA 14= pMA-longlinkerFoka 15= pMA-strep-dig 16= pMA-middlelinker-fokA 18=pMA-split-fokA -&gt;sequencing of clones 1 and 2 of pBAD_Foka <ul> <li>Pellets of: </li> </ul> 19=pMA-fokA 20=pEX-DsbA-strep-dig-split-fokA 21=pEX-strep-fluA(3.10) <ul> <li>digest1: - pBAD-iGEM(Midi 11.10)</li> </ul> - pMA-DsbA-his-dig-split-fokA (Plasmidprep 14.10 Nr5) - pMA-DsbA-strep-dig-split-fokA (Plasmidprep 14.10 Nr4) -10&micro;lDNA -3&micro;l buffer3 -0.5&micro;lBSA(100fold) -14.5&micro;l water -1&micro;l EcoRI -1&micro;l PstI - loaded on 1% agagel <ul> <li>digest2: - pMA-fos-split-fokA (Plasmidprep 14.10 Nr7)</li> </ul> - pEX-fos-split-fokA-K1 -10&micro;lDNA -3&micro;l buffer4 -0.5&micro;lBSA(100fold) -14.5&micro;l water -1&micro;l xbaI -1&micro;l NgoNIV loaded on 1% agagel <ul> <li>digest3: -pMA-DsbA-clone2 (5.10)</li> </ul> -10&micro;lDNA -3&micro;l buffer4 -0.5&micro;lBSA(100fold) -14&micro;l water -1&micro;l xbaI -1.5&micro;l AgeI loaded on 3% agagel -pBAD iGEM -DsbA-his-dig-split-foka -DsbA-strep-dig-split-foka -pMA-fos-dig-split-foka -pEX-fos-split-foka --&gt; Picture <ul> <li>Ligation -pBAD-DsbA-his-dig-split-fokA</li> </ul> -pBAD-DsbA-strep-dig-split-fokA - pMA-fos-dig-split-fokA-DsbA -pEX-fos-split-fokA-DsbA -10&micro;l Qick ligase buffer -1 &micro;l Quick ligase -6&micro;l Insert -3&micro;l Vector <ul> <li> Transformation of the ligation above in X1Blue competent cells</li> </ul> -100&micro;l and rest of each transformation were plated on Amp/Gluc plates <ul> <li>made top agar (0.7% agar) </li> <li>nothing grew on IPTG/Amp plates for test in vivo assay -&gt; made control assays with</li> </ul> - electroporating cotransformed XL1Blue with ssM13DNA and plating them directly to IPTG/Amp cells (no phage precipitation and infection of ER2738) to test electrocompetence - infection of ER2738 with phages from lab phage stock and plating them to IPTG/Amp cells to test plates -&gt;put plates in 37&deg; room overnight <ul> <li>inoclutaion of ER2738 from lab stock and our stock in LB+Tet -&gt;put them on shaker at 26&deg;C </li> <li> XL1 was el. transformed with the ligated AGO-Phagmid library (errore-prone PCR of the AGO-G3P construct into an phagmidvektor with the Tor-A signalling sequence with and without amberstop from yesterday) and plated everything (4 ml)on two big tubs. </li> <li> new PCR for ssDNA from the digested pET39b+ vector to get new target for an additional AGO assay </li> <li>prepaired ELISA to repeat yesterdays AGO-Phage-ELISA tomorrow (because yesterdays results were pretty contradictory -&gt; we probably made a mistake somewhere) </li> </ul> <h3 style="margin-left: 40px;"> 15.10.09, Laura, Caro, Julia, Manu, Timo, Sarah, Hannes, Max, Anika, Christoph <ul> <li>ELISA:</li> </ul> -Blocking: 350&micro;l/well 1% BSA in TBST-EDTA, incubated at 20&deg;C for 2h gently shaking -5x washing with 200&micro;l/well TBST-EDTA -Incubation of phages with oligos for 30min in 55&deg;C waterbath -Incubation of 75&micro;l/well phages for 1h at 20&deg;C gently shaking -5x washing with 200&micro;l/well TBST-EDTA -Antibody: add 75&micro;l/well of anti- M13 antibody (1:1000) -5x washing with 200&micro;l/well TBST-EDTA -Substrate: add 75&micro;l/well 1mg/ml ABTS; measure absorbance immediately and after 3 and 5 min; 405nm <ul> <li>made chemical competent XL1 Blue - laura1:pExFosSplitFoka clone 2</li> </ul> - laura2:pMAFosSplitFoka clone 2 - laura3:pMADsbAHisDigSplitFoka clone 2 - laura4: pMADsbAStrepDigSplitFoka clone 1 <ul> <li>inoculation of - ER2738 from our stock</li> </ul> - XL1 blue from our stock -&gt; in 50 ml LB+Tetracyclin each, put in 37&deg;C -pBAD_Foka clone 1 and 2 from 14.10.09 -pMAFoka clone 1from 04.10.09 -pDsbA clone 2 from 05.10.09 <ul> <li>for in vivo assay - measured pd.7.phage display library concentration new</li> </ul> - mixed 1:10^4, 2*10^6, 2*10^7, 2*10^8 dilutons with ER2738 (OD600 of 0.46) and XL1 blue (OD600 of 0.54) each - let mixtures stand for 10min (infection time) - mixed cells with 3ml topagar each and plated them on 3ml IPTG/XGal plates - put it in 37&deg;C roomin the dark -pExStrepFluASplitFoki from 08.10.09 -&gt; in 5 ml LB+Ampicillin each, put in 37&deg;C <ul> <li> new ssDNA production via pET39b+ digestion product one-primer-pcr </li> <li> elusions from the test panning was given to xl1-&gt; infection-&gt; plated in tet cm -&gt; cells infected with the phagmid should grow-&gt;tomorrow picking clones and determination of the phagmidinsert </li> <li>new AGO BB pcr usind dmso, the gene cut out of the vektor and only the ad-on-tail-primers </li> <li>starter cultures of AGO Aa and AGO Tt in BL21de3 in DYT+Kan </li> <li>starter cultures of pEx-His-Fos-Split-Foka in RV308 in DYT+Amp+1% Glucose </li> </ul> <h3 style="margin-left: 40px;"> 16.10.09, Laura, Caro, Julia, Manu, Timo, Sarah,Isabelle, Hannes, Max, Anika, Christoph <ul> <li>Made 10 L DYT</li> <li>Inoculation of elutions from panning: E1#2 (MnCl2+TBS)n&deg;1-20, E2#1 (MnCl2+TBST)n&deg;1-20, E3#1 (DNAse)n&deg;1-20</li> </ul> <h3 style="margin-left: 40px;"> 17.10.09, Laura, Julia, Manu, Hannes, Gerrit, Christoph <ul> <li>Expression of Ago Tt in Bl21de3 OD measurement </li> </ul> <ul> <li>Dilution from XL1 from OD600 2,8 to OD600 0.07 (MnCl2+TBST)n&deg;1-20, E3#1 (DNAse)n&deg;1-20 wich are phagemids we cultured from elutions of panning from 13.10.09</li> <li>IPTG/X-Gal plates with infected ER2738 was blue, with the infected XL1 blue no blue plaques could be seen -&gt; so infection by phages was positive and lots of blue plaques were built up</li> </ul> <ul> <li> made new in vivo test assay with cotransformated, electrocompetent XL1 blue - transformed M13 ss DNA from 17.08.09 into cells by electroporation at 1.7kV</li> </ul> - 1.5h incubation time on 37&deg;C shaker - 80, 10 and 5&micro;l of electroporated cells were mixed with 180&micro;l XL1 blue and ER2738 with OD 600 of about 0.5 - incubation time of 10 min at room temperature - mixing cells with 3ml topagar, vortexing shortly and plating on IPTG/X-Gal plates - put in 37&deg;C room overnight <ul> <li>control of electrocompetence of XL1 blue by electroporation of pET28a with Kan resistance into XL1 blue and RV respecitvely -&gt; comparison in the end or if colonies will show up at all will show if our cotransformated cells are competent at all - pMAFoka clone 1 from 04.10.09</li> </ul> - pMADsbA clone 2 from 05.10.09 - pBADFoka clone 1 from 15.10.09 - pExStrepFluALongliFoki clone 1 from 08.10.09 <ul> <li>miniprep of all clones picked from the elutions of the test panning to determine the efficiency of the selection </li> <li>new ssDNA via Pet39b+ one primer pcr </li> <li>new infection of XL1 cells with the elution from the test panning-&gt; this time plated on lb agar with tet cm and GLUC (to suppress the expression of the phagmid insert) </li> </ul> <h3 style="margin-left: 40px;"> 18.10.09 Manu <ul> <li>Expression of Ago Aa at 24&deg;C </li> </ul> <h3 style="margin-left: 40px;"> 19.10.09, Laura, Caro, Julia, Manu, Timo, Sarah, Isabel, Hannes, Max, Anika, Christoph <ul> <li> analysis of the geles of the digestion of the clones picked from the elution plates revealed not a single clone containing the ago insert-&gt; possibly the lack of gluc on the plate led to a selection against this insert-&gt; new klones picked from the plate with gluc </li> <li>new ago cleavage assay reveald last saturdays promising results as false. it seems like it was just two conformations of one ssDNA molecule </li> <li>starter cultures of pEx-His-Fos-Split-Fok_a </li> <li>protein purification of GST-FosW with Ni-NTA column - French pressed, centrifuged 15min at 20000rpm, sonicated and filtered</li> </ul> - column was washed with 25mM imidazole and eluted with 250mM imidazole <ul> <li> in vivo assay retried with shorter incubation time </li> </ul> 20.10.09 Manu, Laura, Anika, Julia, Timo, Sarah, Isabel, Max, Gerrit, Christoph, Caro, Hannes <ul> <li> new round of expression with pEx-His-Fos-Split-Fok_a in BL21de3at 24&deg;C </li> </ul>
 * Test expression (15ml) of pBAD_CAT and pBAD_FokA in BL21de3
 * Gelextraction
 * test digests of plasmid preparations from 14.10.09
 * seqencing of
 * inoculation of
 * Minipreps from : E1#2 (MnCl2+TBS)n&deg;1-20, E2#1
 * inoculation of
 * Expression of GST-FosW at 30&deg;C

contact: <a href="mailto:freigem09@googlemail.com">freigem09@googlemail.com</a>