Team:Freiburg bioware/Notebook/July

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01.07.09, 8:30-13:00 Manuel, Hannes

<ul> <li>SDS-Gel:</li> </ul>

<ul> <li>PCR with the purified PCR product from the 30.06.09 as template. Different program: nr. 25 IGEM.AM -&gt; no result. next step should be to do a gradient PCR this afternoon. Just talk to Sven to get the details. </li> <li>inoculated T7+Tt, T7+Aa and BL21+Aa in 3 ml LB-Kan for a new round of expression at 27&deg;C. </li> <li>idea from Sven: do a westernblot on the isolated proteins from the AGO-Expression to find out whether the AGO-protein is present or not. </li> </ul> 01.07.09, 14:00-18:00 Isabel, Laura

<ul> <li>gradient PCR from 60&deg;-70&deg;C with 8 tubes each of vector 258 diluted and undiluted, we put it in the cold room -&gt; gel has to be done tomorrow </li> <li>we made 100ml 1M Tris-HCl pH=6,8 </li> <li>we inoculated 4ml LB with cells from the CAT-vector #258 glycerol stock and put it into the 37&deg;C room </li> <li>to do: -plasmid preparation of the inoculated cells including the CAT vector</li> </ul> <ul style="margin-left: 40px;"> <li>-test-digest of 1,5 &micro;g of the plasmid with EaeI (cutting 3 times, fragments: 2268, 331, 1757 bp)</li> <li>-fetch new dNTPs of the NEB freezer (snd floor)</li> <li>-alterations of PCR-program: only one step but 30 times, with temperature gradient 55-65&deg;C (Sven can help) </li> </ul> 02.07.09, 09:00-18:00 Hannes, 14:00-18:30 Laura

<ul> <li>AGO: expression of Aa and Tt (T7+Tt, T7+Aa, BL21+Aa) at 27&deg;C. samples after 0, 1.5 and 4 hours </li> <li>plasmid preparation of inoculated labvector 258 </li> <li>agarose gel with gradient pcr -&gt; didn't work </li> <li>nanodrop with primer and labvector -&gt; concentrations were good </li> <li>search for a 5 cutter restriction enzyme for the test digest for tomorrow: FOKI! </li> <li>NanoDrop data</li> </ul> 03.07.09, 10:00-13:00 Hannes, Laura, Max, Manuel, Sascha 14:00-18:30 Laura, Hannes, Manuel

<ul> <li>PCR of the CAT-Vektor (258) with new dNTP-Mix and a anealing-gradient from 50&deg;C to 65&deg;C -&gt; bands at every temperature with expected size </li> </ul> <ul> <li>PCR Program </li> </ul>

<ul> <li>SDS-Gel of the samples from yesterday. </li> <li>Laemmli buffer mixed and aliquoted -&gt; -20&deg;C</li> </ul> <img src="http://2009.igem.org/wiki/images/8/88/Freiburg09_030709_Exprssion3001.jpg" name="SDS-Gel of the expression; Lanes: Marker (prestained broad range 7-200 kDa, T7+Aa 1h..2h..4h, T7+Tt 1h..2h..4h, BL21+Aa 1h..2h..4h" height="140" width="225" />

SDS-Gel of the expression; Lanes: Marker (prestained broad range 7-200 kDa, T7+Aa 1h..2h..4h, T7+Tt 1h..2h..4h, BL21+Aa 1h..2h..4h 06.07.09, 09:00-19:00 Hannes, Laura, Caro, Manuel

<ul> <li>we did a agarose gel from the pcr product, cut the band and eluted the DNA. As the concentration only was about 50 ng/&micro;l we did a new gel with 3 times the amount on pcr product (150 &micro;l). We cut the bands, eluted the DNA and got a concentration from about 100 ng/&micro;l. We used both eluted DNA samples for the restriction digest with HindIII and MfeI (4 hours at 37&deg;C). </li> <li>After the restriction digest had been incubated for 4 h (37&deg;C) we purificated it </li> <li>Because of the fact that we eluated with 50ul and not with 30ul (better for the following ligation) we got a concentration of only 87,7ng/ul </li> <li> We heat up the sample to 50&deg;C for 1 min and measured again. Finally we got a concentration of about 100 ng/ul </li> <li> sample Cat gene stored in the -20 &deg;C freezer </li> <li> Inoculation (each 10 ml) for Protein expression and Glycerolstocks </li> <li> Nanodrop data:</li> </ul> 07.07.09, 09:00-14:00 Hannes, Manuel, Sascha, Gerrit; 15:00-19:00 Hannes, Laura, Manuel, Caro

<ul> <li>digestion of the pMal-Vektor with HindIII and MfeI (both from the lab freezer), didn't work. Maybe the MfeI enzyme was bad (expired 2005) </li> <li>started expression of AGO at 20&deg;C. Took samples at 0, 2, 4 and 8 hours centrifuged them, decanted the supernatant and stored the pellets at -20&deg;C in the box for Sascha. </li> <li>inoculation of pMal-Vektor from the lb-amp plates </li> <li>we did lb-chloramphenicol plates with and without IPTG </li> <li>Nanodrop data</li> </ul> 08.07.09, 13:30-14:00 Caro, Sascha; 16:00-21:00 Laura

<ul> <li>new digest of pMAL expression vector and CAT gene with MfeI and HindIII for four hours </li> <li>agarose gel with these two samples -&gt; bands of CAT gene (probably cut) around 700 bp and pMAL vector around 5kb and MalE gene around 1,4kb could be seen, bands of CAT and vector were cut out </li> <li>gel extraction of the bands</li> <ul> <li>-concentration of CAT=22 ng/&micro;l</li> <li>-concentration of pMAL=25 ng/&micro;l</li> </ul> <li>ligation with the quick ligase (total volume of 21&micro;l) </li> <li>transformation of 10 &micro;l of ligated vector with new insert into 100 &micro;l competent cells XBL -&gt; plating of 100 &micro;l on plate with CM and CM+IPTG respectively and 50 &micro;l after centifugation on plate with CM and CM+IPTG respectively -&gt; 37&deg;C room over night -&gt; inoculation can be done tomorrow during the day when the cultures seem to be grown well (after 14 h minimum) </li> <li>Nanodrop data </li> <li>Protein A-280 </li> </ul> <ul> <li> Nucleic acid </li> </ul> 09.07.09, Manuel, Max, Gerrit, Sarah, Sascha, Christoph, Hannes, Isabel, Laura

<ul> <li>Western:</li> </ul> <img src="http://2009.igem.org/wiki/images/6/64/Freiburg09_20090709_1343_anti-His.JPG" name="Westernblot of the AGOexpression; Lanes: empty lane, Tt in BL21 DE3 at 8h and 0h, Aa in BL21 DE3 at 8h and 0h, Tt in T7 at 8h and 0h, Aa in T7 at 8h and 0h 500&micro;l culture each" height="150" width="200" />

Westernblot of the AGOexpression; Lanes: empty lane, Tt in BL21 DE3 at 8h and 0h, Aa in BL21 DE3 at 8h and 0h, Tt in T7 at 8h and 0h, Aa in T7 at 8h and 0h 500&micro;l culture each.

<ul> <li>SDS page, Coomassie staining</li> </ul> <img src="http://2009.igem.org/wiki/images/2/22/Freiburg09_090709_SDSpage018.jpg" name="SDS page of the AGOexpression; Lanes: Protein marker NEB, Aa in BL21 de3 waste fraction, fraction 1, fraction 2, fraction 3, Aa in T7 waste fraction, fraction 1, fraction 2, fraction 3, fraction 4" height="170" width="250" />

SDS page of the AGOexpression; Lanes: Protein marker NEB, Aa in BL21 de3 waste fraction, fraction 1, fraction 2, fraction 3, Aa in T7 waste fraction, fraction 1, fraction 2, fraction 3, fraction 4

<ul> <li> Our AGO proteins are at about 80 kDa. Aa 83,6 kDa and Tt 76,7 kDa </li> <li>His-Tag purification via Ni-NTA column and concentration measurement at the Nanodrop, Volume of peristaltic pump 3,5ml, Suggested expression volume for next time: 2l @ 20&deg; for 8h only; alternat. 28&deg; (see first SDS gel where you can see the band "now" at ~80kDa) quite well. </li> <li>Trafo of Lipocalin (klon2) and Digoxigenin (klon1) in XL1 blue </li> <li>Ligation: Pmal+CAT with Temp.-shift (4-16&deg;C) </li> <li>Trafo of the Ligation (above, 2&micro;l and 5&micro;l) and the old ligation (08.07., 4&micro;l) </li> <li>Trafo of His-Tag, Strep-Tag, Split-Linker and GGGs-Linker in XL1 blue </li> <li>Nanodrop data </li> <li>Protein A-280 </li> </ul> 10.07.09, Manuel, Max, Christoph, Laura

<ul> <li>Trafo of the new Ligation showed only few colonies and the old ligation showed no colonies on CM or CM + IPTG -&gt; inoculation and sequencing of clones on positive plates on monday </li> <li>Trafo of His-Tag, Strep-Tag, Split-Linker showed good results; GGGs-Linker showed no grown colonies -&gt; inoculation on monday </li> <li>SDS-Gel with samples of His-Tag purification via Ni-NTA column and quick staining with comassie </li> </ul> 13.07.09, 09:00-15:00 Uhr Laura, Christoph, Hannes, Caro, Manuel, Sascha

<ul> <li>inoculation of His, Strep, Split-Linker in pMa (Amp) </li> <li>inoculation of the ligation-trafo from last week (CAT) </li> <li>we did 4 SDS-gels (stored at 4&deg;C) </li> </ul> <ul> <li>we prepared 1l of 2YT-medium </li> </ul> 14.07.09, 14:00-21:00 Uhr Gerrit, Julia, Dieter, Max, Imi, Manuel, Christoph

<ul> <li>Plasmid-Preparation of His-Tag, Strep-Tag, Split-Linker, Ligation 9.7. (neu2), Ligation 9.7. (alt), Ligation 8.7. (100 &micro;l and rest) </li> <li>Digestion of FOKI, FOKA with XbaI and PSTI and SPLIT- Linker with SpeI, PstI. </li> <li>Applied on 1% Agarose- Gel and sliced out </li> <li>DNA Gel Extraction with QuiaGen Kit according to the protocol </li> <li>Result: Didnt work (FOKI= 6,28 ng/&micro;l; SPLIT=11,43 ng/&micro;l; FOKA= 5,5 ng/&micro;l) </li> <li>We couldnt find plates with Lipocalin and Digoxigenin, inoculation was not possible --&gt;Transformation has to be done tomorrow! </li> <li>Induced: 14: 15 BL21de3 with 1 ml of 1M IPTG </li> <li>Centrifuged BL21 De3 at 21:50. Pellets in the -20&deg;C freezer </li> <li>Induced T7 at 23:35 with 1 ml of 1M IPTG </li> <li>Nanodrop data</li> </ul> 15.07.09, Hannes, Caro, Laura

<ul> <li>centrifuged the induced Tt/AGO cells at 4&deg;C, 15 min, 4000rpm, discarded the supernatant and put it in the -20&deg;C freezer </li> <li>new digest of Split, FOKa and FOKi -&gt; used wrong tubes (old digest) -&gt; sufficient labelling on the tubes (part, what was done with it, date) necessary!!! </li> <li>for tomorrow: -make trafo of lipocalin, digoxigenin (-&gt; Manu knows where to find them?)</li> <ul> <li>-new digests: Fok, Split, Lipo, Dig, His(?), Strep(?), ligation(?)</li> <li>-purification of AGO with His/Ni column </li> </ul> </ul> 16.07.09,Manuel, Isabel, Sarah, Christoph

<ul> <li>digestion of Split, FOKa and FOKi + gelextraction </li> <li>sequenzing of Lig 8.7. "neu, alt, 100" </li> <li>run agarose gel of the digestion and cut the bands, did a gel extraction and measured the concentration at the NanoDrop. The concentration was again too low, to do a ligation. Put the samples in the -20&deg;C freezer. </li> </ul> <ul> <li>Nanodrop data</li> </ul> 17.07.09,Timo, Gerrit, Sarah, Isabel, Hannes, Christoph

<ul> <li>5 l LBmedia prepared </li> <li> we got a look at the ssDNA of the M13 phage, there is no Fok restriction site. </li> <li> we also made an alignment of the pMAL+CAT vector and the parts we got back from sequencing. the 2.1 clone is faultless. </li> <li>His-Tag purification of Tt and Aa AGOs via Ni-NTA column -&gt; SDS Gel and protein concentration measurement needs to be done on monday (all samples in the -20&deg;C freezer!) </li> </ul> 20.07.09, Gerrit + Imi

<ul> <li>SDS-Gel purification and commassie staining of the purified Aa and Tt AGOs (Samples W2-4, E1-6)</li> </ul> <img src="http://2009.igem.org/wiki/images/3/30/Freiburg09AGO_his_purification004.jpg" name="Aa on the upper gel with M,W2,W3,W4,E3,E1,E4,E2,E6,E5; then Tt on the lower gel with M,W2,W3,W4,E1,E2,E3,E4,E5,E6" height="250" width="230" />

Aa on the upper gel with M,W2,W3,W4,E3,E1,E4,E2,E6,E5; then Tt on the lower gel with M,W2,W3,W4,E1,E2,E3,E4,E5,E6

<ul> <li>Ligation of FokA + Split, FokI + Split </li> <li>Transformations of FokA+Split, FokI+Split, Dig, Lipo, GGGS </li> <li>Transformation of pUC: proliferation of the pUC-vector </li> <li>Inoculation of FokI, FokA, neu1, neu2, Aa vector, Tt vector in LB+Amp in order to create stocks since the cold storage room is out!! </li> <li>Nanodrop data</li> </ul> 20.07.09, Isabel + Sarah

<ul> <li> made TBS buffer (100ml) </li> <li> start preparation of M13 phage ssDNA :overnight breeding of ER2738 </li> </ul> 21.07.09, Isabel, Sarah, Manuel, Max

<ul> <li> made a glycerol stock of ER2738 Tet </li> <li> transformation of ER2738 with M13 phage, breeding and start phage precipitation </li> <li>inoculation of the ligation Fok (inactive)+Split-Linker(2 colonies picked of each) </li> <li>inoculation of Dig and Lipo (2 colonies picked of each) </li> <li>inoculation of Fok (active and inactive), Ligation neu 1 and 2 </li> <li>lb-amp plates casted </li> <li>transformation from yesterday (20.07.) plated on lb-amp again: 100 &micro;l and centrifuged residue </li> </ul> 21.07.09, Laura

<ul> <li>start of documenting the cloning procedures like the design of primers and expressionvector (cloning strategies of genIII and the order of the ligation of the Fok, linker etc has to follow) in the new folder: cloning strategies </li> </ul> 22.07.09, Isabel, Sarah <ul> <li> precipitation of phage M13 particles </li> </ul> 22.07.09, Laura, Christoph

<ul> <li>plasmidpreparation of Ligation (pMAL&amp;CAT) neu 1 &amp; 2, Split linker, Digoxigenin 1 &amp; 2, Lipocalin 1 &amp; 2, Fok/Split Linker 1 &amp; 2 </li> <li>Glycerolstocks of Ligation neu 1 &amp; 2 (Sven did stocks of Foka &amp; Foki, don't know where he put it) </li> <li>digest of His, Strep, Digoxigenin, Lipocalin </li> <li>agarose gel of digests </li> <li>gelextraction of cutted bands -&gt; low concetrations </li> <li>ligation with T4 Ligase of His/Dig, His/Lipo, Strep/Dig, Strep/Lipo -&gt; put it in waterbath for overnight ligation </li> <li>Nanodrop data</li> </ul> 23.07.09, Timo

<ul> <li>preparation of Buffers for anion exchanger </li> <li>picked colonies of the plates: Trafo FokA(21.07.09) and Ligation FokA-Split(20.07.09) and transfered into 3ml LB-Amp </li> <li>check of LB-Amp plates:</li> </ul> Trafo GGGS(1a) 21.07.09 no growth

Trafo GGGS(2a) 21.07.09 no growth

Trafo GGGS(1b) 21.07.09 no growth

Trafo GGGS(2b) 21.07.09 no growth

Trafo Lig.FokA+Split 21.07.09 no growth

Trafo GGGS(1) 20.07.09 no growth

Trafo GGGS(2) 20.07.09 no growth

Ligation FokA+Split 20.07.09 2 colonies

Trafo FokA 21.07.09 some colonies+satellite colonies stored in refrigerator(Premium) 23.07.09, Sarah, Isabel

<ul> <li> complete precipitation of phage M13 particles </li> <li> phage stock in -80&deg;C fridge </li> <li> phage M13 ssDNA precipitation and purification </li> <li> ssDNA in -20&deg;C fridge </li> </ul> 23.07.09, Manu, Laura

<ul> <li>design and order of Gen-III Primers </li> <li>design and order of Short-, Middle- and Long-Linkers for Fok </li> </ul> 23.07.09, Hannes 11:00-17:00

<ul> <li>transformation of ligation from 22.07.09:</li> </ul> - 50&micro;l XBlue + 5&micro;l ligation His-Lipo

- 50&micro;l Xblue + 5&micro;l ligation His-Dig

- 50&micro;l XBlue + 5&micro;l ligation Strep-Dig

- 50&micro;l XBlue + 5&micro;l ligation Strep-Lipo

--&gt; plate out 2 plates: 1. 50&micro;l cells; 2. pellet (5min, 2000rpm) dissolved in 50&micro;l LB

--&gt; 37&deg;C o/n

<ul> <li>transformation to check competent cells:</li> </ul> - 50&micro;l XBlue + 2&micro;l pUC

- 50&micro;l T7 + 2&micro;l pUC

- 50&micro;l BL21 de3 (Novagen) + 2&micro;l pUC

- 50&micro;l RV308 + 2&micro;l pUC

--&gt; plate out 10&micro;l cells + 40&micro;l LB was supposed to be 100 &micro;L cells only, please repeat &amp; plate all cell suspension (Tobias)

<ul> <li>Inoculation for stocks and competent cells:</li> </ul> - 15ml LB+Chloramphenicol with T7 (competent cells)

- 15ml LB+tet with XBlue

- 15ml LB BL21 de3

--&gt; 37&deg;C o/n 24.07.09, Hannes, Christoph, Manuel

<ul> <li> counted colonies from transformation (23.07.09): - BL 21 de3 + pUC: 0 colonies</li> </ul> - RV308 + pUC: 11 colonies

- T7 + pUC: 67 colonies

- XL1blue + pUC: 0 colonies

- ligation of Dig+His, Dig+Strep, FluA+His, FluA+Strep was covered with many colonies

<ul> <li> miniprep of FokA and FokA+split </li> <li> Glycerol stocks (30%) of: - T7</li> </ul> - XBlue

- BL21 de3

--&gt; -80&deg;C

<ul> <li>Nanodrop data</li> </ul> 27.07.09, Imi, Laura

<ul> <li>Inoculation of His-Dig, His-Lipo, Strep-Dig, Strep-Lipo </li> <li>preparation of 100 ml LB+Chloramphenicol and 100ml LB+Tetracyclin </li> <li>Inoclulation of XL blue1, T7 Express in 5 ml LB+antibiotics </li> <li>order of 2 oligos for the M13mp18 ss DNA (there are 4 Fok restriction sites) to cut at two sites resulting in fragments of about 890 and 6360 bp with Fok </li> </ul> 28.07.09, Dieter, Max

<ul> <li>Inoculation of His-Dig, His-Lipo, Strep-Dig, Strep-Lipo, Foki-Split in 5 ml LB+antibiotics (this time on the shaker) </li> <li>preparation of 75 ml LB+Ampicilin </li> <li>Inoclulation of XL blue1, T7 Express in 5 ml LB+antibiotics</li> <li>The Primers for Gene III have arrived </li> </ul> 29.07.09, Isabel, Sarah, Gerrit, Dieter, Laura

<ul> <li>Plasmidpreparation of His-Dig, His-Lipo, Strep-Dig, Strep-Lipo, Foki-Split -&gt; very low concentrations except for His-Lipo and Strep-Dig (just low) -&gt; made a digest with both and pMal-CAT (9.7. clon 2.1) </li> <li>new inoculations of His-Dig, His-Lipo, Strep-Dig, Strep-Lipo, Foki-Split </li> <li>we also made competent cells of T7 and XL Blue (about 50 aliquots each) </li> <li>we poured Amp-plates </li> <li>complementary linker and GenIII-Primer arrived </li> <li>Nanodrop data</li> </ul> 30.07.09, Laura, Manuel, R&uuml;diger, Timo, Hannes, Christoph(2:30am-12am)

<ul> <li> plasmidprep Dig+His, Dig+Strep, FluA+His, FluA+Strep and Foki+Split </li> <li> glycerol stock of Dig+His, Dig+Strep, FluA+His, FluA+Strep and Foki+Split </li> <li> protein purification of Aa protein with Ni-NTA column - pool of fractions E1-E4 and E5-E7 in -80&deg;C (box Aa), washing fractions in cold room on ice</li> <li> digest of Dig+His_clon5, Dig+Strep_clon5, FluA+His_clon6, FluA+Strep_clon5 and pMAL</li> </ul>

<img src="http://2009.igem.org/wiki/images/4/4a/Freiburg092009-07-29_1005.jpg" height="300" width="300" />

<img src="http://2009.igem.org/wiki/images/0/0d/Freiburg092009-07-29_2006.jpg" height="225" width="350" />

<ul> <li> over night-ligation of Dig+His_clon5, Dig+Strep_clon5, FluA+His_clon6, FluA+Strep_clon5 into pMAL </li> <li> transformation of pMAL+CAT into Xblue </li> <li>Nanodrop data</li> </ul> 31.07.09, Labtalk: Max, Gerrit, Isabel, Sarah, Timo, Hannes, Christoph, Sascha, Kristian, Sven, Tobi, Laura Plan for today

<ul> <li>Transformation of ligations His+Dig, Strep+Dig, His+FluA, Strep+FluA in pMAL respectively (Isabel, Sarah) </li> <li>generate an inventory of all stocks and plasmids in our freezers (Isabel, Sarah, Gerrit) </li> <li>PCR with M13-DNA and GenIII-Primers (Max, Laura) </li> <li>PCR with complementary linkers (short, middle and long) to hybridise(Max, Laura) </li> <li>SDS-Gel of purified Argonaut proteins (Hannes) </li> <li>scan gel pictures and upload them into wiki (Christoph) </li> <li>order of new equipment like pipettes, racks etc (Timo) </li> <li>analyse background activity of E. coli (Gerrit) </li> <li>write the project abstract for official wiki (Max) </li> <li>modelling and planing length of oligos(R&uuml;diger) </li> </ul> <b>

Nanodrop data</b>

<ul> <li>Nucleic acid </li> </ul>

<ul> <li>Protein A-280</li> </ul>

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