Team:ETH Zurich/Wet Lab/Protocol

Modelling   Conclusions    Team Page    Organization   Introduction of GFP/RFP into MG1655/MD42 E. coli strains   using the Lambda Red System   Subcloning of GFP/RFP into pCK01 followed by PCR amplification   Day 1: Friday, July 11  <p lang="de-DE" class="western" id="d8on24" style="margin-bottom: 0in;"> digest p18GFP/BB_J14461RFP plasmids and pCK01 2.5 h         <p class="western" id="d8on29" style="margin-bottom: 0in;">25 &micro;L GFP construct/RFP construct/pCK01 plasmid </li> </ul> </li> </ul> <p class="western" id="d8on31" style="margin-bottom: 0in; margin-left: 0.98in; font-family: Arial;">1 &micro;L EcoRI <p class="western" id="d8on33" style="margin-bottom: 0in; margin-left: 0.98in; font-family: Arial;">1 &micro;L PstI <p class="western" id="d8on35" style="margin-bottom: 0in; margin-left: 0.98in; font-family: Arial;">3 &micro;L EcoRI buffer <p class="western" id="d8on37" style="margin-bottom: 0in; margin-left: 0.98in; font-family: Arial;"> <p class="western" id="d8on39" style="margin-bottom: 0in; margin-left: 0.98in; font-family: Arial;">&rarr; 2 h at 37&deg;C <p class="western" id="d8on41" style="margin-bottom: 0in; margin-left: 0.98in; font-family: Arial;">&rarr; pCK01 digest was heat-inactivated for 20 min at 80&deg;C <p class="western" id="d8on43" style="margin-bottom: 0in; margin-left: 0.98in; font-family: Arial;"> <ul id="d8on45" style="font-family: Arial;"> <p lang="de-DE" class="western" id="d8on47" style="margin-bottom: 0in;"> run gel to confirm digest 1 h         <p class="western" id="d8on52" style="margin-bottom: 0in;">0.8 % agarose gel: </li> </ul> </li> </ul> <p class="western" id="d8on54" style="margin-bottom: 0in; margin-left: 0.98in; font-family: Arial;">lane 1 (large!) + 2: 26 &micro;L RFP digest + 6 &micro;L loading buffer (LB) <p lang="de-DE" class="western" id="d8on56" style="margin-bottom: 0in; margin-left: 0.98in; font-family: Arial;">lane 3: 4 &micro;L RFP digest + 2 &micro;L LB <p lang="de-DE" class="western" id="d8on58" style="margin-bottom: 0in; margin-left: 0.98in; font-family: Arial;">lane 4: 2.5 &micro;L &lambda; marker + 2 &micro;L LB <p lang="de-DE" class="western" id="d8on61" style="margin-bottom: 0in; margin-left: 0.98in; font-family: Arial;">lane 5 (large!): 26 &micro;L GFP digest + 6 &micro;L LB <p lang="de-DE" class="western" id="d8on63" style="margin-bottom: 0in; margin-left: 0.98in; font-family: Arial;">lane 6: 4 &micro;L GFP digest + 2 &micro;L LB <p lang="de-DE" class="western" id="d8on65" style="margin-bottom: 0in; margin-left: 0.98in; font-family: Arial;"> <p class="western" id="d8on67" style="margin-bottom: 0in; margin-left: 0.98in; font-family: Arial;">&rarr; run at 90 V for ca. 1 h <p lang="de-DE" class="western" id="d8on69" style="margin-bottom: 0in; margin-left: 0.98in; font-family: Arial;">&rarr; lanes 3/4 and lane 6 cut out and put in EtBr for ca. 30 min <p class="western" id="d8on72" style="margin-bottom: 0in; margin-left: 0.98in; font-family: Arial;"> <p lang="de-DE" class="western" id="d8on74" style="margin-bottom: 0in; margin-left: 0.98in; font-family: Arial;"> pics (4 s exposure) saved under GFP_RFP_restr</i> <p class="western" id="d8on78" style="margin-bottom: 0in; margin-left: 0.98in; font-family: Arial;"> <ul id="d8on80" style="font-family: Arial;"> <p lang="de-DE" class="western" id="d8on82" style="margin-bottom: 0in;"> cut out GFP/RFP bands, recover DNA 1 h         <p lang="de-DE" class="western" id="d8on87" style="margin-bottom: 0in;"> RFP showed 2 bands: one at ca. 1000 bp and one higher </li> </ul> </li> </ul> <p class="western" id="d8on90" style="margin-bottom: 0in; margin-left: 0.75in; text-indent: 0.23in; font-family: Arial;">(ca. 1000 bp + higher expected) <ul id="d8on92" style="font-family: Arial;"> <p lang="de-DE" class="western" id="d8on95" style="margin-bottom: 0in;"> GFP showed 3 bands: one at ca. 1000 bp, one higher and one lower </li> </ul> </ul> <p class="western" id="d8on98" style="margin-bottom: 0in; margin-left: 0.75in; text-indent: 0.23in; font-family: Arial;">(ca. 500, 1000 and 2200 bp expected) <p class="western" id="d8on100" style="margin-bottom: 0in; margin-left: 0.75in; font-family: Arial;"> <p class="western" id="d8on102" style="margin-bottom: 0in; margin-left: 0.98in; font-family: Arial;">&rarr; bands at 1000 bp were cut out (ca. 250 mg) <p class="western" id="d8on104" style="margin-bottom: 0in; font-family: Arial;"> <ul id="d8on106" style="font-family: Arial;"> <p lang="de-DE" class="western" id="d8on109" style="margin-bottom: 0in;"> gel extraction was performed according to protocol: </li> </ul> </ul> <p class="western" id="d8on112" style="margin-bottom: 0in; margin-left: 0.98in; font-family: Arial;">750 &micro;L of buffer QG <p class="western" id="d8on114" style="margin-bottom: 0in; margin-left: 0.98in; font-family: Arial;">250 &micro;L isopropanol <p class="western" id="d8on116" style="margin-bottom: 0in; margin-left: 0.98in; font-family: Arial;">elution with 30 &micro;L EB buffer (wait 1 min before elution!) <p class="western" id="d8on118" style="margin-bottom: 0in; margin-left: 0.75in; font-family: Arial;"> <ul id="d8on120" style="font-family: Arial;"> <p lang="de-DE" class="western" id="d8on122" style="margin-bottom: 0in;"> ligate recovered DNA with digested pCK01 O/N <p class="western" id="d8on127" style="margin-bottom: 0in;">3 &micro;L pCK01 vector </li> </ul> </li> </ul> <p class="western" id="d8on129" style="margin-bottom: 0in; margin-left: 0.98in; font-family: Arial;">1 &micro;L T4 buffer <p class="western" id="d8on131" style="margin-bottom: 0in; margin-left: 0.98in; font-family: Arial;">0.5 &micro;L T4 <p class="western" id="d8on133" style="margin-bottom: 0in; margin-left: 0.98in; font-family: Arial;">5.5 &micro;L GFP insert/RFP insert/ water (control) <p class="western" id="d8on135" style="margin-bottom: 0in; font-family: Arial;"> <p class="western" id="d8on136" style="margin-bottom: 0in; font-family: Arial;">                       &rarr; spin down and put to 4&deg;C O/N <p class="western" id="d8on138" style="margin-bottom: 0in; font-family: Arial;"> <p class="western" id="d8on140" style="margin-bottom: 0in; font-family: Arial;"> Day 2: Saturday, July 12 (5 pm) <ul id="d8on146" style="font-family: Arial;"> <p lang="de-DE" class="western" id="d8on148" style="margin-bottom: 0in;"> transform ligations (3 x) and GFP/RFP/pCK01 plasmids 2 h     </li> </ul> <p lang="de-DE" class="western" id="d8on151" style="margin-bottom: 0in; margin-left: 0.49in; font-family: Arial;"> (to get backups): 6 transformations! <ul id="d8on154" style="font-family: Arial;"> <p class="western" id="d8on157" style="margin-bottom: 0in;">3 ligations transformed: </li> </ul> </ul> <p class="western" id="d8on159" style="margin-bottom: 0in; margin-left: 0.98in; font-family: Arial;">10 &micro;L ligation (GFP/RFP/control) <p class="western" id="d8on161" style="margin-bottom: 0in; margin-left: 0.98in; font-family: Arial;">40 &micro;L competent cells <ul id="d8on163" style="font-family: Arial;"> <p lang="de-DE" class="western" id="d8on166" style="margin-bottom: 0in;"> 3 backup plasmids transformed: </li> </ul> </ul> <p class="western" id="d8on169" style="margin-bottom: 0in; margin-left: 0.98in; font-family: Arial;">2 &micro;L p18GFP/BB_J14461 RFP/pCK01 plasmids <p class="western" id="d8on171" style="margin-bottom: 0in; margin-left: 0.98in; font-family: Arial;">40 &micro;L competent cells <p class="western" id="d8on173" style="margin-bottom: 0in; margin-left: 0.98in; font-family: Arial;"> <p class="western" id="d8on175" style="margin-bottom: 0in; margin-left: 0.98in; font-family: Arial;">&rarr; 20 min on ice <p class="western" id="d8on177" style="margin-bottom: 0in; margin-left: 0.98in; font-family: Arial;">&rarr; 90 sec at 42&deg;C <p class="western" id="d8on179" style="margin-bottom: 0in; margin-left: 0.98in; font-family: Arial;">&rarr; 90 sec on ice <p class="western" id="d8on181" style="margin-bottom: 0in; margin-left: 0.98in; font-family: Arial;">&rarr; 200 &micro;L CVM added <p class="western" id="d8on183" style="margin-bottom: 0in; margin-left: 0.98in; font-family: Arial;">&rarr; 1 h recovery at 37&deg;C and 220 rpm <p class="western" id="d8on185" style="margin-bottom: 0in; margin-left: 0.98in; font-family: Arial;"> <ul id="d8on187" style="font-family: Arial;"> <p lang="de-DE" class="western" id="d8on189" style="margin-bottom: 0in;"> plate out transformations (6 x) and incubate at 37&deg;C O/N </li> </ul> <ul id="d8on192" style="font-family: Arial;"> <p lang="de-DE" class="western" id="d8on195" style="margin-bottom: 0in;"> 3 ligations (GFP-pCK01, RFP-pCK01, control) plated on         </li> </ul> </ul> <p class="western" id="d8on198" style="margin-bottom: 0in; margin-left: 0.75in; text-indent: 0.23in; font-family: Arial;">LB + chloramphenicol (cat) + x-gal agar plates <ul id="d8on200" style="font-family: Arial;"> <p class="western" id="d8on203" style="margin-bottom: 0in;">pCK01 plated on LB + chloramphenicol agar plates, p18GFP and BB_J14461 RFP plated on LB + ampicillin agar plates </li> </ul> </ul> <p class="western" id="d8on205" style="margin-bottom: 0in; font-family: Arial;"> <p class="western" id="d8on207" style="margin-bottom: 0in; margin-left: 0.98in; font-family: Arial;">&rarr; put to 37&deg;C O/N <p class="western" id="d8on209" style="margin-bottom: 0in; font-family: Arial;"> <p lang="de-DE" class="western" id="d8on211" style="margin-bottom: 0in; font-family: Arial;"> Day 3: Sunday, July 13 (5 pm) <ul id="d8on215" style="font-family: Arial;"> <p lang="de-DE" class="western" id="d8on217" style="margin-bottom: 0in;"> pick colonies and inoculate over night cultures: O/N </li> </ul> <p class="western" id="d8on220" style="margin-bottom: 0in; margin-left: 0.49in; font-family: Arial;">1 colony of GFP/RFP/pCK01 and lambda red <p class="western" id="d8on222" style="margin-bottom: 0in; margin-left: 0.49in; font-family: Arial;">+ 2 x 5 of GFP-pCK01/RFP-pCK01 &rarr; 14 cultures <ul id="d8on224" style="font-family: Arial;"> <p class="western" id="d8on227" style="margin-bottom: 0in;">one GFP/RFP/lambda red colony was inoculated in 15 mL-Falcon </li> </ul> </ul> <p class="western" id="d8on229" style="margin-bottom: 0in; margin-left: 0.98in; font-family: Arial;">tubes containing 5 mL LB medium + ampicillin <ul id="d8on231" style="font-family: Arial;"> <p class="western" id="d8on234" style="margin-bottom: 0in;">5 colonies of GFP-pCK01/RFP-pCK01 were inoculated in 5 mL        </li> </ul> </ul> <p class="western" id="d8on236" style="margin-bottom: 0in; margin-left: 0.98in; font-family: Arial;">LB medium + chloramphenicol <p class="western" id="d8on238" style="margin-bottom: 0in; margin-left: 0.98in; font-family: Arial;"> <p class="western" id="d8on240" style="margin-bottom: 0in; margin-left: 0.98in; font-family: Arial;">&rarr; put to 37&deg;C O/N <p class="western" id="d8on242" style="margin-bottom: 0in; font-family: Arial;"> <p lang="de-DE" class="western" id="d8on244" style="margin-bottom: 0in; font-family: Arial;"> Day 4: Monday, July 14 (9 am) <ul id="d8on248" style="font-family: Arial;"> <p lang="de-DE" class="western" id="d8on250" style="margin-bottom: 0in;"> do mini preps of GFP-pCK01, RFP-pCK01 (3 x each) and GFP/RFP/pCK01/lambda red plasmids (10 mini preps): <ul id="ory-"> <p lang="de-DE" class="western" id="bgg20" style="margin-bottom: 0in;"> eluted in 50 &micro;L EB buffer after 1 min incubation at RT         </li> </ul> </li> </ul> <p lang="de-DE" class="western" id="glx3" style="margin-bottom: 0in; font-family: Arial;"> <ul id="glx32" style="font-family: Arial;"> digest and gel to check cloning: <p lang="de-DE" class="western" id="bgg24" style="margin-bottom: 0in;"> GFP/RFP plasmids digested: </li> </ul> </li> </ul> <p lang="de-DE" class="western" id="o2cv" style="margin-bottom: 0in; font-family: Arial;">                   2 &micro;L DNA <p lang="de-DE" class="western" id="d3jm" style="margin-bottom: 0in; font-family: Arial;">                   1 &micro;L EcoRI buffer <p lang="de-DE" class="western" id="uhup0" style="margin-bottom: 0in; font-family: Arial;">                   0.5 &micro;L EcoRI <p lang="de-DE" class="western" id="w:e9" style="margin-bottom: 0in; font-family: Arial;">                   0.5 &micro;L PstI <p lang="de-DE" class="western" id="w:e92" style="margin-bottom: 0in; font-family: Arial;">                   6 &micro;L water <p lang="de-DE" class="western" id="w:e95" style="margin-bottom: 0in; font-family: Arial;"> <ul id="w:e98" style="font-family: Arial;"> <ul id="w:e99"> <li id="w:e910"> GFP-pCK01/RFP-pCK01/pCK01 plasmids digested: </li> </ul> </ul> <p lang="de-DE" class="western" id="w:e914" style="margin-bottom: 0in; font-family: Arial;">                   8 &micro;L DNA <p lang="de-DE" class="western" id="w:e917" style="margin-bottom: 0in; font-family: Arial;">                   1 &micro;L EcoRI buffer <p lang="de-DE" class="western" id="w:e920" style="margin-bottom: 0in; font-family: Arial;">                   0.5 &micro;L EcoRI <p lang="de-DE" class="western" id="w:e923" style="margin-bottom: 0in; font-family: Arial;">                   0.5 &micro;L PstI <p lang="de-DE" class="western" id="ok:9" style="margin-bottom: 0in; font-family: Arial;"> <p lang="de-DE" class="western" id="ok:92" style="margin-bottom: 0in; font-family: Arial;">                   &rarr; put to 37&deg;C for 2 h  <p lang="de-DE" class="western" id="ok:94" style="margin-bottom: 0in; font-family: Arial;"> <ul id="w:e929" style="font-family: Arial;"> <ul id="w:e930"> <p lang="de-DE" class="western" id="eeps0" style="margin-bottom: 0in;"> run on 0.8 % gel: </li> </ul> </ul> <p lang="de-DE" class="western" id="eeps3" style="margin-bottom: 0in; font-family: Arial;">                   add 2 &micro;L LB  <p lang="de-DE" class="western" id="ok:96" style="margin-bottom: 0in; font-family: Arial;">                    load 10 &micro;L of each <p lang="de-DE" class="western" id="ok:99" style="margin-bottom: 0in; font-family: Arial;">                   run at 100 V  <p lang="de-DE" class="western" id="it4j" style="margin-bottom: 0in; font-family: Arial;">                    take pictures <p lang="de-DE" class="western" id="it4j2" style="margin-bottom: 0in; font-family: Arial;"> <p lang="de-DE" class="western" id="it4j5" style="margin-bottom: 0in; font-family: Arial;">                   &rarr; hardly any DNA visible <p lang="de-DE" class="western" id="h0gk" style="margin-bottom: 0in; font-family: Arial;"> <ul id="h0gk1" style="font-family: Arial;"> Inoculation of an additional 3 colonies of GFP-pCK01 and RFP-pCK01 inoculated in 5 mL chloramphenicol containing LB medium </li> incubated at 37&deg;C O/N </li> </ul> </li> </ul> <p lang="de-DE" class="western" id="zqji" style="margin-bottom: 0in; font-family: Arial;"> <p lang="de-DE" class="western" id="gs2b" style="margin-bottom: 0in; font-family: Arial;"> <u id="s.pp">Day 5: Tuesday, July 15 <ul id="gs2b1" style="font-family: Arial;"> GFP-pCK01, RFP-pCK01 (3 x each) and GFP/RFP/pCK01/lambda red plasmids were run on 0.8% gel without digestion </li> </ul> <p lang="de-DE" class="western" id="vjte" style="margin-bottom: 0in; font-family: Arial;">         &rarr; double bands (several colonies picked?!) <ul id="vjte1" style="font-family: Arial;"> Mini prep of 2 x 3 GFP-pCK01/RFP-pCK01 cultures <ul id="s.pp0"> mini preps (2 x 2) were performed and run on 0.8% gel </li> </ul> </li> </ul> <p lang="de-DE" class="western" id="s.pp1" style="margin-bottom: 0in; font-family: Arial;">                   &rarr; 3/4 positive <p lang="de-DE" class="western" id="gs2b5" style="margin-bottom: 0in; font-family: Arial;"> <p lang="de-DE" class="western" id="zqji1" style="margin-bottom: 0in; font-family: Arial;"> Day 6: Wednesday, July 16 <ul id="z7cs0" style="font-family: Arial;"> do PCR of GFP-pCK01 and RFP-pCK01 constructs using the ordered primers primers were dissolved in xxx &micro;L/xxx &micro;L ddH2O (forward/reverse) </li> Master-Mix (MM): </li> </ul> </li> </ul> <p lang="de-DE" class="western" id="jkap0" style="margin-bottom: 0in; font-family: Arial;">                   40 &micro;L 5 x Taq buffer <p lang="de-DE" class="western" id="jkap3" style="margin-bottom: 0in; font-family: Arial;">                   32 &micro;L 25 mM MgCl2 <p lang="de-DE" class="western" id="jkap6" style="margin-bottom: 0in; font-family: Arial;">                   2 &micro;L ATP <p lang="de-DE" class="western" id="jkap9" style="margin-bottom: 0in; font-family: Arial;">                   2 &micro;L GTP <p lang="de-DE" class="western" id="jkap12" style="margin-bottom: 0in; font-family: Arial;">                   2 &micro;L CTP <p lang="de-DE" class="western" id="jkap15" style="margin-bottom: 0in; font-family: Arial;">                   2 &micro;L TTP <p lang="de-DE" class="western" id="jkap18" style="margin-bottom: 0in; font-family: Arial;">                   8 &micro;L forward primer (100 &micro;M) <p lang="de-DE" class="western" id="jkap20" style="margin-bottom: 0in; font-family: Arial;">                   8 &micro;L reverse primer (100 &micro;M) <p lang="de-DE" class="western" id="ukkm" style="margin-bottom: 0in; font-family: Arial;">                   8 &micro;L Taq polymerase <p lang="de-DE" class="western" id="ukkm1" style="margin-bottom: 0in; font-family: Arial;">                   272 &micro;L ddH2O <ul id="x9bi" style="font-family: Arial;"> 3 different amounts of GFP-pCK01 (no. 2, 15.07.08)/RFP-pCK01 (no. 1, 15.07.08) template were used: </li> </ul> </ul> <p lang="de-DE" class="western" id="pnxg" style="margin-bottom: 0in; font-family: Arial;">                   49 &micro;L MM + 1 &micro;L template <p lang="de-DE" class="western" id="pnxg1" style="margin-bottom: 0in; font-family: Arial;">                   48 &micro;L MM + 2 &micro;L template <p lang="de-DE" class="western" id="pnxg3" style="margin-bottom: 0in; font-family: Arial;">                   46 &micro;L MM + 4 &micro;L template <p lang="de-DE" class="western" id="pu9j" style="margin-bottom: 0in; font-family: Arial;"> <p lang="de-DE" class="western" id="pu9j1" style="margin-bottom: 0in; font-family: Arial;">                   &rarr; 6 PCRs <p lang="de-DE" class="western" id="eex8" style="margin-bottom: 0in; font-family: Arial;"> <ul id="eex81" style="font-family: Arial;"> PCR program: </li> </ul> </ul> <p lang="de-DE" class="western" id="eex87" style="margin-bottom: 0in; font-family: Arial;">                   5 min 95&deg;C <p lang="de-DE" class="western" id="sw_1" style="margin-bottom: 0in; font-family: Arial;"> <p lang="de-DE" class="western" id="sw_11" style="margin-bottom: 0in; font-family: Arial;"><span lang="en-US" id="sw_12">                   35 x  <p lang="de-DE" class="western" id="eex810" style="margin-bottom: 0in; font-family: Arial;">                    1:50 min 95&deg;C <p lang="de-DE" class="western" id="wn-n" style="margin-bottom: 0in; font-family: Arial;">                   1:50 min 50&deg;C <p lang="de-DE" class="western" id="wn-n1" style="margin-bottom: 0in; font-family: Arial;">                   2 min 72&deg;C <p lang="de-DE" class="western" id="wn-n3" style="margin-bottom: 0in; font-family: Arial;"> <p lang="de-DE" class="western" id="pu9j3" style="margin-bottom: 0in; font-family: Arial;">                   5 min 72&deg;C <p lang="de-DE" class="western" id="sw_13" style="margin-bottom: 0in; font-family: Arial;"><span lang="en-US" id="sw_14">                   4&deg;C after finish; running time: ca. 4:30 h  <p lang="de-DE" class="western" id="j6qk" style="margin-bottom: 0in; font-family: Arial;"> <ul id="j6qk1" style="font-family: Arial;"> check and purify PCR product on gel 10 &micro;L LB added to all 6 PCR products </li> 6 &micro;L loaded for checking </li> rest (ca. 50 &micro;L) loaded in large pockets for extraction </li> 6 &micro;L lambda marker loaded (3 &micro;L marker + 3 &micro;L LB) </li> run at 100 V on 0.8% gel </li> </ul> </li> </ul> <p lang="de-DE" class="western" id="pdk1" style="margin-bottom: 0in; font-family: Arial;"> &rarr; no bands, PCR needs to be repeated! <u id="bkk_">Day 7: Thursday, July 17 <ul id="r4on" style="font-family: Arial;"> repitition of PCR with changed protocol <ul id="s5.f"> <li id="s5.f0"> 2.5 &micro;L high fidelity buffer (with MgCl2, no.2) </li> </ul> </li> </ul> 1 &micro;L primer up (10 &micro;M!) 1 &micro;L primer down (10 &micro;M!) 0.5 &micro;L dNTPs (10 &micro;M of each dATP, dGTP, dCTP, dTTP; is aliquoted) 0.5/1 &micro;L template (GFP-cat 15.07.08 no. 3, RFP-cat 15.07.08 no. 1) <div lang="de-DE" class="western" id="h_-c" style="margin-bottom: 0in; font-family: Arial;">                    0.5 &micro;L high fidelity polymerase 19 &micro;L ddH2O <p lang="de-DE" class="western" id="kofs0" style="margin-bottom: 0in; font-family: Arial;"> &rarr; 4 PCRs a 25 &micro;L <ul id="vzeu0" style="font-family: Arial;"> program: </li> </ul> </ul> 5 min 94&deg;C <div lang="de-DE" class="western" id="ci_-" style="margin-bottom: 0in; font-family: Arial;"> <div lang="de-DE" class="western" id="ci_-0" style="margin-bottom: 0in; font-family: Arial;">                   1 min 95&deg;C <div lang="de-DE" class="western" id="ci_-1" style="margin-bottom: 0in; font-family: Arial;">                   1 min 55&deg;C <div lang="de-DE" class="western" id="ci_-2" style="margin-bottom: 0in; font-family: Arial;">                   2 min 72&deg;C <div lang="de-DE" class="western" id="ci_-3" style="margin-bottom: 0in; font-family: Arial;">                   &rarr; 25 x <div lang="de-DE" class="western" id="ci_-4" style="margin-bottom: 0in; font-family: Arial;"> <div lang="de-DE" class="western" id="ci_-5" style="margin-bottom: 0in; font-family: Arial;">                   10 min 72&deg;C <div lang="de-DE" class="western" id="ci_-6" style="margin-bottom: 0in; font-family: Arial;">                   4&deg;C till end <div lang="de-DE" class="western" id="ci_-7" style="margin-bottom: 0in; font-family: Arial;"> <ul id="ci_-8" style="font-family: Arial;"> <li id="ci_-9"> <div lang="de-DE" class="western" id="ci_-10" style="margin-bottom: 0in;">0.8% gel to check and extract PCR product <ul id="ci_-11"> <li id="ci_-12"> <div lang="de-DE" class="western" id="ci_-13" style="margin-bottom: 0in;">5 &micro;L LB was added to each </li> <li id="ci_-14"> <div lang="de-DE" class="western" id="ci_-15" style="margin-bottom: 0in;">run at 100 V        </li> </ul> </li> </ul> &rarr; bands at 2500 kb for all 4 samples <ul id="w1tm0" style="font-family: Arial;"> gel extraction gel extraction was performed using 1100 &micro;L buffer QG and 350 &micro;L isopropanol </li> samples were eluted with 30 &micro;L EB buffer (wait 1 min before centrifugation!) </li> </ul> </li> </ul> <p class="western" id="x9bi7" style="margin-bottom: 0in; font-family: Arial;"> Day 8: Friday, July 18 <ul id="fvei1" style="font-family: Arial;"> run extracted PCR products on 0.8 % gel to check extraction and size Agarose gel electrophoresis of PCR products (2&micro;l 100 bp Fermentas DNA Marker; 3&micro;l PCR product GFP 0.5, GFP 1, RFP 0.5, RFP 1) </li> PCR product is larger than 1kb, yield exceeds 30ng/ul </li> </ul> </li> </ul> &rarr; clear bands for all 4 PCR products!  <div id="tc_5" style="padding: 1em 0pt; text-align: left;"><img alt="" id="khah" style="width: 389px; height: 211px;" src="http://docs.google.com/File?id=ddsp7zr4_1fkdvbchq_b" /> &rarr; ready for transformation into lambda red-transformed, competent MG1655/MD42 strains (see below) <p class="western" id="d8on275" style="margin-bottom: 0in; font-family: Arial;"> <p class="western" id="d8on277" style="margin-bottom: 0in; font-family: Arial;"> Preparation of competent MG1655 and MD42 strains for introduction of GFP/RFP constructs</b> <p class="western" id="d8on282" style="margin-bottom: 0in; font-family: Arial;"> <p lang="de-DE" class="western" id="d8on284" style="margin-bottom: 0in; font-family: Arial;"> Day 1: Thursday, July 10 <p class="western" id="d8on290" style="margin-bottom: 0in; font-family: Arial;">plate out glycerol stocks of MG1655 and MD42 done <p id="fvei8" style="font-family: Arial;"> <ul id="d8on292" style="font-family: Arial;"> <p class="western" id="d8on294" style="margin-bottom: 0in;">scrape off some of icy glycerol stock and plate on LB agar </li> </ul> <p class="western" id="fvei9" style="margin-bottom: 0in; margin-left: 0.98in; font-family: Arial;"> <p class="western" id="d8on298" style="margin-bottom: 0in; margin-left: 0.98in; font-family: Arial;">&rarr; incubate at 37&deg;C O/N <p class="western" id="d8on300" style="margin-bottom: 0in; font-family: Arial;"> <p lang="de-DE" class="western" id="d8on302" style="margin-bottom: 0in; font-family: Arial;"> Day 2: Friday, July 11 <ul id="d8on306" style="font-family: Arial;"> <p class="western" id="d8on308" style="margin-bottom: 0in;">put colonies to 4&deg;C <p class="western" id="d8on312" style="margin-bottom: 0in;">colonies have grown and are ready to be picked! </li> </ul> </li> </ul> <p class="western" id="d8on314" style="margin-bottom: 0in; font-family: Arial;"> <p class="western" id="d8on316" style="margin-bottom: 0in; font-family: Arial;"> Day 3: Monday, July 14 <ul id="d8on319" style="font-family: Arial;"> <p lang="de-DE" class="western" id="d8on321" style="margin-bottom: 0in;"> prepare competent MG1655/MDS42 cells <ul id="jy.z"> <li id="jy.z0"> <p lang="de-DE" class="western" id="jy.z1" style="margin-bottom: 0in;"> 2 colonies of MG1655 and MDS42 each inoculated in 5 mL LB medium (5 pm) </li> <p lang="de-DE" class="western" id="nrek0" style="margin-bottom: 0in;">incubated at 37&deg;C O/N </li> </ul> </li> </ul> <p lang="de-DE" class="western" id="nspg2" style="margin-bottom: 0in; font-family: Arial;"> Day 4: Tuesday, July 15 <ul id="did10" style="font-family: Arial;"> prepare competent MG1655/MDS42 cells (continued) 50 mL LB medium were inoculated with 500 &micro;L over night cultures and grown to OD(600) 0.488 (MG1655) and 0.503 (MDS42) </li> </ul> </li> </ul> <p lang="de-DE" class="western" id="pap3" style="margin-bottom: 0in; font-family: Arial;">                    (MG1655 grew faster</i> and was stored on ice while MDS42 was still growing) <ul id="okg2" style="font-family: Arial;"> cultures were transfered to 50 mL Falcon tube (on ice!) and spun down for 10 min at 2500 rpm and 4&deg;C </li> discard supernatant </li> pellet was resuspended in 50 mL buffer 1 (pre-cooled!) </li> store on ice for 90 min </li> spin 10 min at 2500 rpm and 4&deg;C </li> discard supernatant </li> resuspend pellet in 2 mL ice cold buffer 2 </li> 200 &micro;L each were filled in 1.5 mL Eppis and stored at -80&deg;C </li> </ul> </ul> <p lang="de-DE" class="western" id="q5e4" style="margin-bottom: 0in; font-family: Arial;"> <ul id="q5e43" style="font-family: Arial;"> transform competent MG1655/MDS42 cells with lambda red constructs (pKD46) 2 &micro;L pKD46 were transformed into 50 &micro;L of competent MG1655/MDS42 according to the protocol </li> heat shock at 42&deg;C </li> 200 &micro;L CVM added </li> recovery at 30&deg;C </li> plated on ampicillin containing agar plates at 30&deg;C (!) O/N </li> </ul> </li> </ul> <p lang="de-DE" class="western" id="y26e4" style="margin-bottom: 0in; font-family: Arial;"> <p lang="de-DE" class="western" id="y26e5" style="margin-bottom: 0in; font-family: Arial;"> <u id="lr8_">Day 5: Wednesday, July 16 <ul id="y26e6" style="font-family: Arial;"> <p lang="de-DE" class="western" id="d8on332" style="margin-bottom: 0in;"> prepare competent lambda red MG1655/MD42 pKD46 cells <p lang="de-DE" class="western" id="pbfk1" style="margin-bottom: 0in;"> inoculate 2 colonies of each MG1655/MDS42 pKD46 in 5 mL ampicillin containing LB medium </li> <p lang="de-DE" class="western" id="l94e0" style="margin-bottom: 0in;"> incubate at 30&deg;C O/N at 200 rpm </li> </ul> </li> </ul> <p lang="de-DE" class="western" id="l94e3" style="margin-bottom: 0in; font-family: Arial;"> <p lang="de-DE" class="western" id="l94e6" style="margin-bottom: 0in; font-family: Arial;"> Day 6: Thursday, July 17 <ul id="c5b90" style="font-family: Arial;"> prepare competent lambda red MG1655/MD42 pKD46 cells (continued) protocol as described on day 4 (with MG1655/MD42 pKD46</i> cells!) </li> grown to OD(600) of 0.458 (MG1655)/0.536 (MDS42) </li> </ul> </li> </ul> Again, MG1655 grew faster</i>! <ul id="ru9b16" style="font-family: Arial;"> cells were stored on ice for 20 min before 1st centrifugation </li> aliquoted in 2 x 11 Eppis a 200 &micro;L and stored at -80&deg;C </li> </ul> </ul> <p lang="de-DE" class="western" id="vjq6" style="margin-bottom: 0in; font-family: Arial;"> Day 7: Friday, July 18 <p lang="de-DE" class="western" id="vjq63" style="margin-bottom: 0in; font-family: Arial;"> <ul id="lrt6" style="font-family: Arial;"> <p class="western" id="vjq610" style="margin-bottom: 0in;">transform MG1655/MD42 pKD46 cells with GFP-pCK01/RFP-pCK01 PCR products (see above) temperature shock transformation of MG1655/MD42 pKD46 cells with GFP-pCK01/RFP-pCK01 PCR products according to protocol: </li> </ul> </li> </ul> For the transformation, 50ul of competent cells and 2ul of PCR product (amounting to approximately 100 ng) were used and streaked onto LB-Cat plates. Incubation at 30&deg;C for 12 hours to three days did not yield any colonies. &rarr; Transformation has failed and needs to be repeated. Day 8: Tuesday, July 22 Repetition of PCR product transformations: 1 &micro;L/5 &micro;L/10 &micro;L of GFP-cat PCR product (0.5) and 2 &micro;L pCK01 (control) were transformed in 45 &micro;L MG1655 + pKD46 cells </li> 1 &micro;L/5 &micro;L/10 &micro;L of RFP-cat PCR product (0.5) and 2 &micro;L pCK01 (control) were transformed in 45 &micro;L MDS42 + pKD46 cells </li> 10 min on ice, heat shock at 42&deg;C, recovery with 200 &micro;L CVM at 37&deg;C, plated on chloramphenicol containing agar plates, incubation at 37&deg;C O/N </li> </ul> </li> </ul> Day 9: Wednesday, July 23 <ul id="a_kn"> <li id="a_kn0">Picking colonies for O/N cultures of MG1655/MD42 pKD46 in 5mL LB medium with ampicillin.</li> </ul> <u id="a_kn3"> Day 9: Thursday, July 24     Production of competent cells with induction of lambda-red-recombination syste</b>m:</b><u id="hpa-2"> Inoculation with 500 uL O/N culture of MG1655/MD42 pKD46 in 50mLB medium with ampicillin and 500 uL 1M Arabinose. \</li> growth until OD(600nm) = ~ 0.4 in 30C! shaker (MG1655 grew slightly faster and were put on ice 15 min earlier).</li> cells on ice for 30 min</li> centrifuge cells for 10 min, 2500 rpm, 4&deg;C, discard supernatant</li> wash cells twice with 10 mL ice-cold ddH2O and twice with 10 mL 10% Glycerol.</li> cells resuspended in 500 &micro;L 10 % glzcerol and aliquoted to 50 &micro;L per Eppi</li> <li id="sg:t">stored at -80&deg;C</li> </ul> Transformation with insert through electroporation: </b> add to 50 uL competent cells (MG1655/MD42 pKD46) 5 uL/10 uL insert (GFP-cat-1/RFP-cat-1 from PCR of 17.7.08) </li> <li id="pfn_">put into ice-cold electroporation-tube</li> <li id="pfn_0">pulse with 1.25 kV, add immediately 1 mL LB</li> <li id="la:7">put into a sterile epi and recover in 37C shaker for 1 h</li> <li id="fpg-">centrifuge 1 min, 12 000 rpm, discard supernatant, resuspend in residual medium</li> <li id="fpg-0">plate on Cat-plates and incubate O/N at 37C</li> </ul> Day 10: Friday, July 25 Result: Transformation has failed and needs to be repeated.  Day 11: Monday, 28 July   Repetition of transformation through electroporation incl. control</b>  <ul id="v.ra"> <li id="v.ra0">add to 50 ul competent cells (MG1655/MD42) from 24. 6.08 insert (1 uL pUCP26, contains tet-resistence or 3ul RFP-cat/GFP-cat)</li> electroshock at 1.25kV, add immediately 1mL LB + 10 uL 1M arabinose</b></li> recover in 37C shaker for 3 hours</b></li> plate 50% and incubate O/N at 37C</li> let 50% of cells on the bench in 1ml LB O/N</li> </ul>  Day 12: Tuesday, 29 July Results from electroporation from 28.7.08</b> control strains (transformation with pUCP26) are grown: => electroporation has been successful</li> GFP/RFP-cat insertion failed again => integration of insert into chromosome has failed</li> </ul>  <span id="ajoy" style="background-color: rgb(234, 153, 153);">   Test of Insertion of GFP/RFP into Target gene TnaA</b>  <ul id="yypw2" style="background-color: rgb(234, 153, 153);"> one colony found on plate from 24.7.08, MD42 transformed with 10uL RFP-cat </li> inoculation of colony in 5 ml LB with Chloramphenicol, and incubation in 37C shaker (start 11am - about 3/4pm until OD = 2)</li> Tryptophan-Color-Test: positive ! addition of Kovac's Regent for indoles stayed yellow, and didnt turn red. Therefore TnaA is inactive, probably by insertion of RFP-cat.</li> Fluorescence microscopy: clearly moving particles were visible under LM, some red dots under FM</li> </ul>  <ul id="yypw10" style="background-color: rgb(234, 153, 153);"> Inoculation of O/N culture in 5 ml LB with Chloramphenicol. </li> Incubation of Backup plates from the one colony found in 37C oven.</li> </ul> Growth experiment of MG1655/MD42 in LB</b> Inoculate MG1655/MD42 in 5 ml LB 37 shaker over night.  <br id="iw:v1" /> <u id="iw:v2">Day 13: Wednesday, 30 July<br id="lw_i" /> <br id="lw_i0" /> <b id="lw_i2" style="background-color: rgb(234, 153, 153);">   Test of Insertion of GFP/RFP into Target gene TnaA</b> Indol-test with O/N culture from 29.7.08 OD = 4.605 again positiv: Medium stayed yellow upon addition of Kovac's Regent for indoles.</li> to do: FL after 2h in freezer</li> to do: Primer design for PCR to verify insertion</li> </ul> <u id="lw_i3"><br id="iw:v4" /> Growth experiment of MG1655/MD42 in LB</b> Inoculation of 100ml media with O/N culture to OD(600) = 0.05: 2.358 mL MG1655 (preculture with OD(600) = 2.12) and 1.976 mL MD42 (preculture with OD(600) = 2.53)</li> <li id="b1-:">OD measurements:</li> </ul> <img width="450" height="320" alt="" id="mqen" src="http://docs.google.com/File?id=dhf5kqn7_5cx2xg4d6_b" /> <div id="dr_:" style="padding: 1em 0pt; text-align: left;"> <img alt="" id="e_n9" style="width: 450px; height: 320px;" src="http://docs.google.com/File?id=dhf5kqn7_8cm2vzghd_b" /> => max growth rate: MG1655: u = 1.57 1/h</li> MDS42: u = 1.10 1/h</li> </ul> </li> => max OD600 MG1655: 4.55</li> MDS42: 4.55</li> </ul> </li> </ul> Digestion of Insert and pKD46</b><br id="b-_2" /> digestion of 8.5 uL pKD46(MG)/pKD46(MD) in 1 uL EcoR I Buffer and 0.5 uL EcoR I</li> </ul> digestion of 3 uL RFP-cat/GFP-cat in 5.5 uL ddH2O and 1 uL Buffer 0 and 0.5 uL Pst I</li> <li id="b-_20">Digestion for 2h in 37&deg;Oven</li> </ul> load with 2 uL LB, from left to right: lambda marker, pkD42 MG, pKD42 MD, GFP MG, RFP MD.</li> </ul> <img width="137" height="333" alt="" id="w4fx" style="margin: 1em 0pt 0pt 1em; float: left;" src="http://docs.google.com/File?id=dhf5kqn7_3ff2dk4ct_b" /> <img width="138" height="332" alt="" id="i5cz" src="http://docs.google.com/File?id=dhf5kqn7_4c5wm22fb_b" /> <u id="y.q12">Appendix <img border="0" id="jgfp3" title="GeneRuler&trade; &amp; O'GeneRuler&trade; 100 bp DNA Ladders" style="width: 300px; height: 434px;" alt="" src="http://www.fermentas.com/catalog/electrophoresis/images/generulersm0241.jpg" /> <td bgcolor="#000000"> <td bgcolor="#000000" width="100%"> http://www4.clustrmaps.com/stats/maps-no_clusters/2008.igem.org-Team-ETH_Zurich-thumb.jpg