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__NOTOC__ {{:Team:LMU-Munich/Templates/Page Header}} =ApoControl Notebook= =='''Contents'''== {|class="wikitable centered" border="2" rules="rows" width="100%" style="border-color:white;" |- ! Week !! colspan="7" |Days |- | |style="text-align:center"|Monday |style="text-align:center"|Tuesday |style="text-align:center"|Wednesday |style="text-align:center"|Thursday |style="text-align:center"|Friday |style="text-align:center"|Saturday |style="text-align:center"|Sunday |- |style="text-align:center"| 31 |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#8-02-2010|8-02-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#8-03-2010|8-03-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#8-04-2010|8-04-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#8-05-2010|8-05-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#8-06-2010|8-06-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#8-07-2010|8-07-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#8-08-2010|8-08-2010]] |- |style="text-align:center"| 32 |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#8-09-2010|8-09-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#8-10-2010|8-10-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#8-11-2010|8-11-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#8-12-2010|8-12-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#8-13-2010|8-13-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#8-14-2010|8-14-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#8-15-2010|8-15-2010]] |- |style="text-align:center"| 33 |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#8-16-2010|8-16-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#8-17-2010|8-17-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#8-18-2010|8-18-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#8-19-2010|8-19-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#8-20-2010|8-20-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#8-21-2010|8-21-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#8-22-2010|8-22-2010]] |- |style="text-align:center"| 34 |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#8-23-2010|8-23-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#8-24-2010|8-24-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#8-25-2010|8-25-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#8-26-2010|8-26-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#8-27-2010|8-27-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#8-28-2010|8-28-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#8-29-2010|8-29-2010]] |- |style="text-align:center"| 35 |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#8-30-2010|8-30-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#8-31-2010|8-31-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#9-01-2010|9-01-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#9-02-2010|9-02-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#9-03-2010|9-03-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#9-04-2010|9-04-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#9-05-2010|9-05-2010]] |- |style="text-align:center"| 36 |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#9-06-2010|9-06-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#9-07-2010|9-07-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#9-08-2010|9-08-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#9-09-2010|9-09-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#9-10-2010|9-10-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#9-11-2010|9-11-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#9-12-2010|9-12-2010]] |- |style="text-align:center"| 37 |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#9-13-2010|9-13-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#9-14-2010|9-14-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#9-15-2010|9-15-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#9-16-2010|9-16-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#9-17-2010|9-17-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#9-18-2010|9-18-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#9-19-2010|9-19-2010]] |- |style="text-align:center"| 38 |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#9-20-2010|9-20-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#9-21-2010|9-21-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#9-22-2010|9-22-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#9-23-2010|9-23-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#9-24-2010|9-24-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#9-25-2010|9-25-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#9-26-2010|9-26-2010]] |- |style="text-align:center"| 39 |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#9-27-2010|9-27-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#9-28-2010|9-28-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#9-29-2010|9-29-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#9-30-2010|9-30-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-01-2010|10-01-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-02-2010|10-02-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-03-2010|10-03-2010]] |- |style="text-align:center"| 40 |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-04-2010|10-04-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-05-2010|10-05-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-05-2010|10-06-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-07-2010|10-07-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-08-2010|10-08-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-09-2010|10-09-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-10-2010|10-10-2010]] |- |style="text-align:center"| 41 |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-11-2010|10-11-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-12-2010|10-12-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-13-2010|10-13-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-14-2010|10-14-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-15-2010|10-15-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-16-2010|10-16-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-17-2010|10-17-2010]] |- |style="text-align:center"| 42 |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-18-2010|10-18-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-19-2010|10-19-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-20-2010|10-20-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-21-2010|10-21-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-22-2010|10-22-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-23-2010|10-23-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-24-2010|10-24-2010]] |- |style="text-align:center"| 43 |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-25-2010|10-25-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-26-2010|10-26-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-27-2010|10-27-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-28-2010|10-28-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-29-2010|10-29-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-30-2010|10-30-2010]] |style="text-align:center"| [[Team:LMU-Munich/Notebook/Apoptosis#10-31-2010|10-31-2010]] |- |} == 8-10-2010 == <font color="#009933">Transforming competent cells</font> - eGFP Biobrick: BBa_I714891 SDY_eGFP (Kanamycin) - TEV recogn N Degron SF3 = pDS7 (Ampicillin) - TEV p14 recogn = 190-6 (Ampicillin) -> Protocol: ([[Team:LMU-Munich/Notebook/Protocols/3_Transformation|3 Transformation]]) - We added 2 µl DNA - We plated out 200 µl <font color="#009933">Plasmid Isolation</font> - CMV-Promoter Biobrick: BBa_J52034 -> Protocol:([[Team:LMU-Munich/Notebook/Protocols/4_Plasmid_extraction_from_cells|4 Plasmid extraction from cells]]) - Prepared overnight culture, measured concentration of DNA -> Poor results -> thrown away == 8-11-2010 == <font color="#009933">New Plasmid Extraction</font> - CMV-Promoter Biobrick: BBa_J52034 -> Protocol: ([[Team:LMU-Munich/Notebook/Protocols/4_Plasmid_extraction_from_cells|4 Plasmid extraction from cells]]) - Plasmid concentration: 143ng/µl <font color="#009933">Prepared overnight culture of eGFP BBa_I714891</font> - 3 ml LB-Media + 4 µl Kanamycin - Inoculated iangeimpft) with 1 colony of BBa_I714891 -> 37°C <font color="#009933">Prepared overnight culture of 190-6 and pDS7 and eGFP (BBa_I714891) in falcons</font> - for 190-6 and pDS7: 10µl Ampicillin + 10 ml LB-Media + colony of plate - for eGFP: 13,3 µl Kanamycin + 10 ml LB-Media + 1 colony of plate <font color="#009933">Restriction digestion of CMV-Promoter BBa_J52034 with EcoRI and PstI</font> {| |H₂O_{ddest, sterile} |10,3 µl |- |RE10 + Buffer H |2,0 µl |- |acetylated BSA (18ng/µl) |0,2 µl |- |DNA (0,143µg/µl) |6,0 µl |} -> mixed - plus: EcoRI (10µg/µl): 0,5 µl resp. PstI (10µg/µl): 0,5 µl - incubated at room temperature from 12:10 to 15:00, 1 hour at 37°C, 2 hours at 60°C - frozen at -20°C <font color="#009933">Prepare new/fresh overnight culture of CMV-Promoter Biobrick: BBa_J52034</font> - 1 ml of "old" culture + 3 ml LB-Media + 4 µl Kanamycin -> 37°C == 8-12-2010 == <font color="#009933">Plasmid Extraction of pDS7, eGFP, 190-6</font> -> Protocol: ([[Team:LMU-Munich/Notebook/Protocols/4_Plasmid_extraction_from_cells|4 Plasmid extraktion from cells]]) - pDS7 (458ng/µl), eGFP (55ng/µl), 190-6 (193ng/µl) <font color="#009933">Restriction digest of pDS7, eGFP, 190-6</font> - with EcoRI and PstI in buffer H (for testing DNA is correct) -> Protocol: ([[Team:LMU-Munich/Notebook/Protocols/5_Restriction_digest|5 Restriction digest]]) - 10µg DNA: pDS7 (2µl), eGFP (15µl), 190-6 (10µl) <font color="#009933">Plate colonies for plasmid extraction</font> - CMV (Kanamycin), eGFP (Kanamycin), pDS7 (Ampicillin), 190-6 (Ampicillin)) - PhiC31o plated on Ampicillin-Agar, stored at 37°C <font color="#009933">50% Glycerol made</font> - for PhiC31o glycerol stock (produced later) == 8-13-2010 == [[Image:GelverdauCMVpDS71906eGFP.jpg|thumb|right|Gelfoto from the EcoR1 and Pst1 Restrictiondigest of 190-6, eGFP, pDS7 and CMV]] <font color="#009933">Inoculate CMV into LB medium with ampicillin</font> - CMV (BBa_J52034) from 10.8.2010 inoculated into LB medium with ampicillin, as falsly inoculated in Kanamycin <font color="#009933">Agarosegelelectrophoresis with digestions</font> ->Protocol ([[Team:LMU-Munich/Notebook/Protocols/11_Agarose_gel_electrophoresis|11 Agarose gel electrophoresis]]) - Agarosegelelectrophoresis with the digestions (CMV, eGFP, pDS7, 190-6), 125V for 30 minutes and then for 20 minutes; - expected DNA bands: 190-6 (4840bp, 1903bp), pDS7 (8027bp, 6bp), CMV (654 bp (Insert), 2079bp (Plasmid)), eGFP (720bp (Insert), 2750bp (Plasmid)) - Correct DNA bands for 190-6 (~4800bp, ~1900bp, ~6700bp (undigested plasmid)) and eGFP (~2000bp (Plasmid), ~750 bp (Insert)); CMV probably not digested (two bands; one probably normal, one supercoiled) and pDS7 not clear <font color="#009933">Restriction digest from CMV and pDS7</font> -> Protocol ([[Team:LMU-Munich/Notebook/Protocols/5_Restriction_digest|5 Restriction digest]]) - Restriction digest from CMV (EcoR1, Pst1; 6µl DNA, buffer H) and pDS7 (EcoR1, Spe1; 2µl DNA, buffer B) <font color="#009933">Agarosegelectrophoresis with digestions</font> ->Protocol ([[Team:LMU-Munich/Notebook/Protocols/11_Agarose_gel_electrophoresis|11 Agarose gel electrophoresis]]) - Agarosegelelectorphoresis for 30 minutes, 150V - Expected DNA bands: CMV see above, pDS7 (3647bp, 3369bp, 1011bp, 6bp) - false DNA bands CMV (~1200 bp, ~2000 bp) and pDS7 (~8000bp two bands, ~1100 bp); required to isolate a new colony for these two Plasmidextractions <font color="#009933">Plated CMV on Ampicllin-Agar</font> - Plated the colony from CMV (BBa_J52034) for Plasmidextraction (Ampicillin), as falsly plated on Kanamycin == 8-14-2010 == weekend == 8-15-2010 == weekend == 8-16-2010 == <font color="#009933">Planting colonies</font> - transfer 1 ml PhiC31o culture to new LB medium + Amp, 37°C - pick up CMV and pDS7 colonies from plates and transfer to LB medium+Amp, 37°C <font color="#009933">Plasmid Extraction of PhiC31o</font> ->Protocol ([[Team:LMU-Munich/Notebook/Protocols/4_Plasmid_extraction_from_cells|4 Plasmid extraktion from cells]]) - plasmid extraction of PhiC310 ->27,5ng/µl DNA and second plasmid extraction of PhiC310 (i. o. to get more DNA); first eluation-step with first eluation-extraction -> 60ng/µl DNA <font color="#009933">Restriction digest</font> ->Protocol ([[Team:LMU-Munich/Notebook/Protocols/5_Restriction_digest|5 Restriction digest]]) - restriction digest of PhiC310 with EcoR1 and Spe1 {| |H₂O_{ddest, sterile} |0 µl |- |Buffer B |2,0 µl |- |BSA (1:10) |2 µl |- |DNA (0,06µg/µl) |15,0 µl |- |EcoR1 |0,5 µl |- |Spe1 |0,5µl |} restriction digest in the thermo cycler (program "Verdau", see protocol) <font color="#009933">Handling primers after arrival (1,2,3,4,5,6,11,12)</font> ->Protocol ([[Team:LMU-Munich/Notebook/Protocols/9_Handling_primers|9 Handling primers]]) <font color="#009933">PCR preparations</font> - 10mM dNTP mix made from 100 mM dATP, dGTP, dCTP, dTTP by taking 100µl of each and adding 600µl H ₂ O <font color="#009933">PCR 1 and 6</font> - PCR of the tet inducible CMV minimal promotor out of prevTRE (=PCR 1 with Primer 1 and 2) and SV40PA out of pcDNA3 (=PCR 6 with Primer 11 and 12) ->Protocol ([[Team:LMU-Munich/Notebook/Protocols/10_PCR_with_Pfu|10 PCR with Pfu]]) Mixture: {| |- | |pTRERev (0,15µg/µl) |pcDNA3 (0,6 µg/µl) |- |Primer |2*2,5µl (P1+P2) |" (P11+P12) |- |300ng template | 0,5µl | 2µl |- |10x Buffer Pfu |5µl |" |- |dNTP Mix |1µl |" |- |Pfu Polymerase (3u/µl) |0,5µl |" |- |H₂O |40,5µl |39µl |- |summ |52,5µl |52,5µl |} Programme: {| |- |Denaturation |95°C |2min |- |30 times: |Denaturation |95°C |1min |- | |Annealing |45°C |30sec |- | |Extension |73°C |2min |- |Final Extension |73°C |5min |- |Soak (end) |12°C |infinite |} <font color="#009933">Glycerolstock of PhiC31o</font> - Glycerolstock of the colony of PhiC31o for the plasmidextraction {| |- |bacterial culture |800µl |- |Glycerol (50%) |500µl |} == 8-17-2010 == [[Image:17.8.2010 PCR6.jpg|thumb|right|Agarose gel electrophoresis of PCR6 which shows that PCR6 is about 200bp]] <font color="#009933">Plate CMV and pDS7 colonies on Ampicillin-Agar</font> - colonies for plasmidextraction of CMV and pDS7 plated on Ampicillinplates <font color="#009933">Plasmid Extraction of CMV and pDS7</font> - plasmidextraction of CMV (2,5ng/µl) and pDS7 (10ng/µl) the A260/A280 value was 1.333, which means that it was 90% Protein and only 10% DNA (should be 1,8); new plasmidextraction needed <font color="#009933">new overnight cultures of CMV and pDS7 for a new plasmidextraction made</font> <font color="#009933">Agarose gel electrophoresis</font> -> Protocol ([[Team:LMU-Munich/Notebook/Protocols/11_Agarose_gel_electrophoresis|11 Agarose gel electrophoresis]]) - Agarose gel electrophoresis of the restriction digest of PhiC31o and PCR 1 and 6 - the right bands found for PhiC31o (~2900,~2400,~250) - the right band found for PCR1 (~450) - no band found for PCR6; new electrophoresis needed with more DNA loaded <html> <table> <tr> <td> <img src="https://static.igem.org/mediawiki/2010/b/ba/17_8_10_1_apo.jpg" width="265" height="159" border=0 /> </td> <td> <img src="https://static.igem.org/mediawiki/2010/c/c6/17_8_10_2_apo.jpg" width="259" height="153" border=0 /> </td> </tr> <tr> <td> Agarose gel electrophoresis of (from left to right) PhiC31o, PCR1 and PCR6 </td> <td> Agarose gel electrophoresis of (from left to right) PhiC31o, PCR1 and PCR6 which shows that PCR1 is between 250 and 500 bp </td> </tr> </table> </html> - new agarose gel electrophoresis from PCR6 with 5µl DNA instead of 3µl (image not yet shown) - the right band found for PCR6 (~200) <font color="#009933">New overnight cultures of CMV and pDS7</font> - the overnight colonies didn't grow; new colonies (CMV and pDS7) picked from plate and inoculated in LB Ampicillin <font color="#009933">PCR purification of PCR 1 and 6</font> -> Protocol ([[Team:LMU-Munich/Notebook/Protocols/12_Gel_extraction_or_PCR_Clean_up|12 Gel extraction or PCR Clean up]]) - DNA concentration of the PCR 1 and 6 products measured: PCR1: 410ng/µl (A260/A280=1.253) PCR6: 568ng/µl (A260/A280=1.275) - PCR Purification with Promega Kit -> PCR1: 230ng/µl (A260/A280=1.769) -> PCR6: 37.5ng/µl (A260/A280=1.667) == 8-18-2010 == [[Image:18_8_10_apo.jpg|thumb|right|Agarose gel electrophoresis of (from left to right) CMV and pDS7 showing the right bands for pDS7]] <font color="#009933">Plasmid Extraction of CMV and pDS7</font> -> Protocol ([[Team:LMU-Munich/Notebook/Protocols/4_Plasmid_extraction_from_cells|4 Plasmid extraction from cells]]) - Plasmid extraction of CMV (97.5ng/µl; A260/A280=1.857) and pDS7 (212ng/µl; A260/A280=1.848) <font color="#009933">Restriction digestion</font> -> Protocol ([[Team:LMU-Munich/Notebook/Protocols/5_Restriction_digest|5 Restriction digestion]]) - Restriction digestion of CMV (EcoR1 + Pst1; 10µl DNA, buffer H) and pDS7 (EcoR1 + Spe1; 5µl DNA, buffer B) -> expected DNA bands: CMV: 2079bp (plasmid) + 654bp (Insert); pDS7: 7022bp + 1011bp <font color="#009933">Agarose Gel electrophoresis of digested CMV and pDS7</font> -> Protocol ([[Team:LMU-Munich/Notebook/Protocols/11_Agarose_gel_electrophoresis|11 Agarorse gel electrophoresis]]) -> right DNA bands for pDS7 (~7000bp, ~1000bp) -> false DNA bands for CMV - Starting PCR 2a and 2b (replication and mutagenesis of pDS7): 3 µl DNA and 50°C Annealing Temperatur (other same as 8-16-2010) == 8-19-2010 == <font color="#009933">Agarose gel electrophoresis of PCR 2a and 2b</font> -> Protocol ([[Team:LMU-Munich/Notebook/Protocols/11_Agarose_gel_electrophoresis|11 Agarose gel electrophoresis]]) (150V, 30min) {| |[[Image:19 8 10 apo.jpg|170px|Agarose gel electrophoresis of (from left to right) PCR2a and PCR2b]] |- |Agarose gel electrophoresis of (from left to right) PCR2a and PCR2b |} -> the right bands for PCR2a (~300bp) and PCR2b (~700bp) - New agarose gel electrophoresis with all of the PCR product for gel extraction (150V, 30min) <font color="#009933">Gel extraction of the DNA from PCR2a and PCR2b</font> -> Protocol ([[Team:LMU-Munich/Notebook/Protocols/12_Gel_extraction_or_PCR_Clean_up|12 Gel extraction or PCR Clean up]]) - DNA concentration measured; problem with nanodrop as too low concentration; lyophille used to reduce volume - DNA concentration measured again: PCR2a: 70ng/µl A260/A280=1.647; PCR2b: 45ng/µl A260/A280=1.5 <font color="#009933">PCR 3 (joining PCR of 2a and 2b)</font> - PCR3 (the joining PCR of PCR2a and 2b; Joining of the TEVrecogn-N-Degron-SF3 part) done: 1.3 µl of PCR2a and 4.7 µl of PCR2b makes 300ng of a 1:1 solution of both to be joined DNA parts. Annealing temperature: 50°C -> Protocol ([[Team:LMU-Munich/Notebook/Protocols/10_PCR_with_Pfu|10 PCR with Pfu]]) == 8-20-2010 == <font color="#009933">Agarose gel electrophoresis of PCR3</font> [[Image:GelverdauPCR3-1.jpg|400px|Gelfoto from PCR3]] left column: marker; rightmost column: PCR3 -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]] (150V, 30min) -> expected band: ~1000bp -> false band: ~500bp - probable reason: mini photometre was influenced by gel extraction chemicals, therefore it measured false DNA concentrations and false template masses were calculated -> New 2a and 2b PCR <font color="#009933">New PCR (2a and 2b)</font> -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/10 PCR with Pfu| 10 PCR with Pfu]] (see 8-18-2010, but 35,5µl water) == 8-21-2010 == weekend == 8-22-2010 == weekend == 8-23-2010 == <font color="#009933">Agarose gel electrophoresis of PCR 2b</font> -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]] - expected band: 700bp -> no band shown on gel -> new PCR 2b <font color="#009933">PCR 2b</font> - start PCR 2b with PCR 2b from 8-13-10 as template ( 1:20 and 1:100 diluted; 1µl) -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/10 PCR with Pfu| 10 PCR with Pfu]] - annealing temperature: 50°C; amount of water: 37,5µl <font color="#009933">Agarose gel electrophoresis of PCR 2b 1:20 and 1:100</font> -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]] - expected bands: each ~ 700bp - false bands: ~ 200bp -> new PCR with 2ng, 5ng, 10ng template pDS7 <font color="#009933">PCR 2b with 2ng, 5ng, 10ng template pDS7</font> - pDS7 1:100 diluted(-> 2,1 ng/µl) Mixture: {| |- | |2ng |5ng |10ng |- |Primer |2*2,5µl (P5+P6) |2*2,5µl (P5+P6) |2*2,5µl (P5+P6) |- |10x Buffer Pfu |5µl |5µl |5µl |- |dNTP Mix |1µl |1µl |1µl |- |template |pDS7 (dil.) |1µl |2,5µl |5µl |- |Pfu Polymerase (3u/µl) |0,5µl |0,5µl |0,5µl |- |DMSO |1,25µl |1,25µl |1,25µl |- |H₂O |33,25µl |30,25µl |25,25µl |- |sum | | | |} -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/10 PCR with Pfu| 10 PCR with Pfu]] <font color="#009933">PCR 2a gel extraction</font> - Quiagen kit (QuiaexII) -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/14 QIAEX II gel extraction| 14 QIAEX II gel extraction]] <font color="#009933">Start 3 CMV overnight cultures</font> == 8-24-2010 == <font color="#009933">agarose gel electrophoresis of PCR 2b</font> -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]] [[Image:24_8_10_apo3.jpg|thumb|right|Agarose gel electrophoresis of (from left to right) PCR2b (2ng (cut out), 10ng, 5ng template) showing the right bands for 2ng, 5ng template]] - expected bands: right bands with 2ng and 5ng template (~700bp), no band with 10ng template <font color="#009933">CMV plasmid extraction</font> -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/4 Plasmid extraction from cells|4 Plasmid extraction from cells]] Plasmid extractionof 3 different overnight cultures. - results: * 52,5 ng/µl A260/A280= 1.312 * 133 ng/µl A260/A280= 1.710 * 80 ng/µl A260/A280= 1.600 <font color="#009933">CMV restriction digestion</font> -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/5 Restriction digest|5 Restriction digestion]] - CMV restriction digest: EcoRI, PstI, buffer H * 19µl, H₂O : 0µl * 6µl, H₂O : 9.5µl * 12.5µl, H₂O : 3µl <font color="#009933">PCR 2b gel extraction</font> - PCR2b was gel extracted (with Qiagen gel extraction kit), 17.5 ng/µl a260/A280= 1.750 -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/14 QIAEX II gel extraction| 14 QIAEX II gel extraction]] <font color="#009933">PCR 3 (fusion of 2a and 2b)</font> - PCR3: conducted again at 52°C annealing temperature * 10.5 ng (from PCR2b) 0.6µl * 4.5 ng (from PCR2a) 0.9µl (1:10 diluted) ::{| |- |PCR2a |0.9 µl |- |PCR2b |0.6 µl |- |primer3 |2.5 µl |- |primer6 |2.5 µl |- |dNTPs |1 µl |- |Pfu |0.5 µl |- |10xbuffer |5 µl |- |H₂O |37 µl |} -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/10 PCR with Pfu| 10 PCR with Pfu]] <font color="#009933">agarose gel electrophoresis of CMV digestion</font> - agarose gel electrophoresis (150V, 25 min) of the CMV digestion -> bands are wrong again ( ~ 1200bp, 2000bp) == 8-25-2010 == <font color="#009933">Agarose gel electrophorese of PCR 3</font> -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]] - expected band: ~1000bp - false band: ~400bp <font color="#009933">Plasmid extraction of ccdB tet and ccdB strep</font> Plasmid extraction of pSB1C3 with BBa_P1010 -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/4 Plasmid extraction from cells|4 Plasmid extraction from cells]] - results: {| |- |ccdB tet: |50ng/µl; |A260/A280= 1,818 |- |} <font color="#009933">Plate ccdB amp, cam, tet</font> - plate ccdB with ampicilline, chloramphenicol, tetracycline resistance on LB agar with appropiate antibiotic. <font color="#009933">Overnight culture of ccdB kan</font> - Overnight culture of ccdB with kanamycine resistance in LB medium with kanamycine <font color="#009933">PCR 7a, 7b, 9, 10</font> ->Protocol: [[Team:LMU-Munich/Notebook/Protocols/10 PCR with Pfu| 10 PCR with Pfu]] {| |- |PCR nr. |template |concentration |dilution |primer |- |7a |190-6 |~200ng/µl |1:100 |13,14 |- |7b |190-6 |~200ng/µl |1:100 |15,16 |- |9 |eGFP |55ng/µl |1:25 |20,21 |- |10 |PhiC31o |20ng/µl |1:10 |22,23 |- |} Mixture {| |- |template (~2ng) |1µl |- |Pfu |0,5µl |- |Primer *2 |2,5µl *2 |- |10x buffer |5µl |- |dNTP Mix |1µl |- |H₂O |37,5µl |- |sum |50µl |- |} Standard PCR; annealing temperature: 60°C == 8-26-2010 == <font color="#009933">Agarose gelelectrophoresis of PCR 7a, 7b, 9, 10</font> ->Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]] - 150V, 25min {| |- |PCR nr. |expected bands |result |- |7a |850bp |no band |- |7b |402bp |false band (200bp) |- |9 |808bp |no band |- |10 |1888bp |no band |- |} <font color="#009933">Plasmid extraction of ccdB kan</font> -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/4 Plasmid extraction from cells|4 Plasmid extraction from cells]] -result: concentration: 25ng/µl; A260/A280= 2,0 <font color="#009933">New PCR 7a, 7b, 9, 10</font> Mixture {| |- |template (~2ng) |1µl |- |Pfu |0,5µl |- |Primer *2 |2,5µl *2 |- |10x buffer |5µl |- |dNTP Mix |1µl |- |DMSO |1,25µl |- |H₂O |36,25µl |- |sum |50µl |- |} Program: gradient PCR, 42-69°C annealing temp. ->Protocol:[[Team:LMU-Munich/Notebook/Protocols/10 PCR with Pfu| 10 PCR with Pfu]] <font color="#009933">Overnight culture of ccdB amp, tet, cam</font> Inoculate one colony each in 5ml medium with approptraite antibiotic. == 8-27-2010 == <font color="#009933">Agarose gel electrophoresis of PCR 7a, 7b, 9, 10</font> ->Protocol:[[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]] 150V, 25min, 75mA [[Image: 27-8-10-1.jpg|400px|gel electrophoresis of new PCR 7a,7b, 9, 10]] from left to right: 7a, 7b, 9, 10, Marker {| |- |PCR nr. |expected bands | result |- |7a |850bp |no band |- |7b |402bp |right band (~400bp)+ false band (~150bp) |- |9 |808bp |false band (~200bp) |- |10 |1888bp |right band (~1900bp)+false band (~500bp) |- |} <font color="#009933">Plasmid extraktion of ccdB amp, tet, cam</font> ->Protocol: [[Team:LMU-Munich/Notebook/Protocols/4 Plasmid extraction from cells|4 Plasmid extraction from cells]] results: {| |Plasmid |concentration |A260/A280 |- |ccdB amp |57,5 ng/µl |1,917 |- |ccdB cam |70,0 ng/µl |1,867 |- |ccdB tet |50,0 ng/µl |1,818 |- |} <font color="#009933">New PCR 7a, 9</font> Mixture: - 2ng template: see 26-8-10 - 4ng template: see 26-8-10, but 2µl template and 35,25µl water -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/10 PCR with Pfu| 10 PCR with Pfu]] <font color="#009933">Restriction digestion of ccdB amp, kan, cam, tet</font> -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/5 Restriction digest|5 Restriction digestion]] - only 90min 37°C incubation - EcoRI, PstI, Buffer H {| |template |volume |mass |- |ccdB amp |16µl |930ng |- |ccdB cam |14,3µl |1µg |- |ccdB tet |16µl |800ng |- |ccdB kan |16µl |400ng |- |} <font color="#009933">Agarose gelelectrophoresis of PCR 7a, 9, ccdB restriction digestion</font> 150v, 25min, 75mA -> Protocol:[[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]] [[Image: 27 8 10 ccdb.jpg|400px|gel electrophoresis of new PCR 7a, 9, ccdb]] results: - PCR7a, 9: false band at 200bp - ccdB: each digestion leads to a right band with ~ 650bp == 8-28-2010 == weekend == 8-29-2010 == weekend == 8-30-2010 == <font color="#009933">New PCR 7a and 9</font> Mixture {| |- |template (~4ng) |2µl |- |Pfu |0,5µl |- |Primer *2 |2,5µl *2 |- |10x buffer |5µl |- |dNTP Mix |1µl |- |DMSO |1,25µl |- |H₂O |35,25µl |- |sum |50µl |- |} -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/10 PCR with Pfu| 10 PCR with Pfu]] PCR program PCR 7: Annealing Temperature 60°C - 25 x 1 min Annealing time and 5x 1,30 min Annealing time PCR 9: Annealing Temperature 55°C - 25 x 1 min Annealing time and 5x 1,30 min Annealing time <font color="#009933">Gel extraction of PCR 7b, 10</font> ->Protocol: [[Team:LMU-Munich/Notebook/Protocols/14 QIAEX II gel extraction| 14 QIAEX II gel extraction]] results: {| |PCR nr. |concentration |A260/A280 |- |7b |10 ng/µl |2,0 |- |10 |17,5 ng/µl |1,4 |- |} <font color="#009933">Agarose gel electrophoresis of new PCR 7a, 9</font> -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]] 150V, 25min - results: - 7a: no band shown - 9: false band (~200bp) <font color="#009933">New PCR 3</font> -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/10 PCR with Pfu| 10 PCR with Pfu]] ::{| |- |PCR2a |0.9 µl |- |PCR2b |0.6 µl |- |dNTPs |1 µl |- |Pfu |0.5 µl |- |10xbuffer |5 µl |- |DMSO |1,25µl |- |H₂O |36,75 µl |- |sum |45µl |} - new method: standard PCR without primers (10 cycles, 56°C annealing temp.) - then add 2,5µl of primer 3 and 6 - 30 cycles standard PCR (54°C annealing temp.) == 8-31-2010 == [[Image:31 8 10 apo1.jpg|thumb|right|Gel photo of (left to right) PCR4a(2.5ng template), PCR4a(5ng template),PCR4b(2.5ng template), PCR4b(5ng template), PCR3(Pfu), PCR3(Phusion)]] <font color="#009933">Agarose gel electrophoresis of PCR 4a, PCR4b, PCR3(Pfu), PCR3(Phusion)</font> -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]] 150V, 25min - results: - PCR4a(2.5ng template), PCR4a(5ng template),PCR4b(2.5ng template), PCR4b(5ng template), PCR3(Pfu): no band shown - PCR3 (Phusion): right band (~1000bp) <font color="#009933">New PCR PCR4a, PCR4b, PCR7a, PCR9</font> -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/10 PCR with Pfu| 10 PCR with Pfu]] PCR mixture for PCR4a, PCR4b ::{| |- |template |37.25 µl (200ng) |- |dNTPs |1 µl |- |Pfu |0.5 µl |- |10xbuffer |5 µl |- |DMSO |1,25µl |- |sum |50µl |} Standard PCR program with annealing temperature PCR4a: 51.1°C, PCR4b: 48.5°C. -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/15 PCR with Phusion| 15 PCR with Phusion]] PCR mixture for PCR7a, PCR9 ::{| |- |template |4 µl (8ng) |- |dNTPs |1 µl |- |Phusion |0.5 µl |- |5xbuffer |10 µl |- |DMSO |1,25µl |- |H₂O |28.25 µl |- |sum |50µl |} PCR program: Phu62 ::{| |- |98°C |1 min |- |98°C |10 sec |- |62°C |20 sec |- |73°C |30 sec |- |return to step 2 for 29 cycles | |- |73°C |10 min |- |12°C |forever |} [[Image:31 8 10 apo2.jpg|thumb|right|Gel photo of (left to right) PCR3, ladder, PCR7a, empty, PCR9]] <font color="#009933">Agarose gel electrophoresis of PCR 3, PCR7a, PCR9 for gel extraction</font> -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]] 120V, 30min - results: - PCR3; right band (~1000bp) and side-product - PCR7a: no band - PCR9: right band (~800bp) and side-product <font color="#009933">Gel extraction of the DNA from PCR3 and PCR9</font> -> Protocol ([[Team:LMU-Munich/Notebook/Protocols/12_Gel_extraction_or_PCR_Clean_up|12 Gel extraction or PCR Clean up]]) results: PCR 9: 22,5ng/µl; A260/A280=1,8 PCR 3: 22,5ng/µl; A260/A280=2,25 <font color="#009933">New PCR 4a, 4b, 7a with DreamTaq</font> -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/16 PCR with DreamTaq| 16 PCR with DreamTaq]] PCR mixture for PCR7a ::{| |- |template |5 µl (10ng) |- |dNTPs |5 µl |- |DreamTaq |0.33 µl |- |10xbuffer |5 µl |- |DMSO |1,25µl |- |H₂O |28.5 µl |- |sum |50µl |} Primers for PCR 7a: 13,14 Annealing temp: 60°C PCR mixture for PCR4a,4b ::{| |- |template |36,5 µl (180ng HeLa cDNA) |- |dNTPs |5 µl |- |DreamTaq |0.33 µl |- |10xbuffer |5 µl |- |DMSO |1,25µl |- |sum |50µl |} PCR 4a Primers for PCR 4a: 7,8 Primers for PCR 4b: 9,10 Annealing temp: 50°C PCR program: ::{| |- |95°C |1 min |- |95°C |30 sec |- |50/60°C |30 sec |- |72°C |1 min (1kb/min) |- |return to step 2 for 29 cycles | |- |72°C |10 min |- |12°C |forever |} == 9-01-2010 == <font color="#009933">Agarose gel electrophoresis of PCR4a, PCR4b, PCR7 (DreamTaq)</font> -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]] 150V, 25min results: no product <font color="#009933">New PCR 4a, 4b with DreamTaq, Pfu, with concentration gradient and touch-down PCR </font> -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/16 PCR with DreamTaq| 16 PCR with DreamTaq]]; [[Team:LMU-Munich/Notebook/Protocols/10 PCR with Pfu| 10 PCR with Pfu]] PCR mixture for DreamTaq ::{| |- |Concentration |Low |Middle |High |- |template |5 µl (1:1000) |31.75µl (1:1000) |2µl (1:10) |- |dNTPs | |5 µl | |- |DreamTaq | |0.33 µl | |- |10xbuffer | |5 µl | |- |DMSO | |1,25µl | |- |H₂O |26.75 µl |0 |29.75 |- |sum | |50µl | |} PCR mixture for Pfu ::{| |- |Concentration |Low |Middle |High |- |template |5 µl (1:1000) |37.25µl (1:1000) |2µl (1:10) |- |dNTPs | |1 µl | |- |Pfu | |0.5 µl | |- |10xbuffer | |5 µl | |- |DMSO | |1,25µl | |- |H₂O |32.25 µl |0 |35.25 µl |- |sum | |50µl | |} Primers for PCR 4a: 7,8; PCR 4b: 9,10 -> Protocol: Thermal cycler program: [[Team:LMU-Munich/Notebook/Protocols/17 Special thermal cycler programs/Touch down|Touch down]] <font color="#009933">Agarose gel electrophoresis of PCR4a, PCR4b</font> -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]] 150V, 25min [[image: 9.1.10apo.jpg|400px|gel 9-1-10]] from left to right: 4a: P1, P2, P3, D1, D2, D3; 4b: P1, P2, P3, D1, D2, D3 key: "P"= PCR with Pfu "D"= PCR with DreamTaq "1"= low template concentration "2"= middle template concentration "3"= high template concentration expected bands: *4a: 330bp -> P2 and P3 show right bands and "primer clouds"(?) *4b: 376bp -> P1, P2, P3 show right bands and "primer clouds" (?) == 9-02-2010 == <font color="#009933">Agarose gel electrophorese of PCR 4a P2, 4b P2</font> -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]] - 120V, 45min, 1,5% Agarose gel [[Image:2 9 10 apo.jpg|400px|Agarose gel electrophoresis of (from left to right) PCR4aP2, Marker and PCR4bP2]] Agarose gel electrophoresis of (from left to right) PCR4aP2, Marker and PCR4bP2 - Cut out bands at ~~ 350bp and extract <font color="#009933">PCR Agarose gel extraction</font> -> Protocol:[[Team:LMU-Munich/Notebook/Protocols/14 QIAEX II gel extraction| 14 QIAEX II gel extraction]] results: {| |PCR nr. |concentration |A260/A280 |- |4aP2 |12,5 ng/µl |1,67 |- |4bP2 |72,5 ng/µl |1,53 |- |} <font color="#009933">New PCR 7a with Pfu and Phusion</font> template: 190-6, Primer 13,14 Mixture with Pfu {| |- |template (~2ng) |1µl |- |Pfu |0,5µl |- |Primer *2 |2,5µl *2 |- |10x buffer |5µl |- |dNTP Mix |1µl |- |DMSO |1,25µl |- |H₂O |36,25µl |- |sum |50µl |- |} -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/10 PCR with Pfu| 10 PCR with Pfu]] PCR mixture with Phusion ::{| |- |template (~2ng) |1,0 µl |- |dNTPs |1 µl |- |Phusion |0.5 µl |- |5xbuffer |10 µl |- |DMSO |1,25µl |- |H₂O |31,25µl |- |sum |50µl |} -> Protocol:[[Team:LMU-Munich/Notebook/Protocols/15 PCR with Phusion| 15 PCR with Phusion]] <font color="#009933">New PCR 4a, 4b with Pfu</font> -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/10 PCR with Pfu| 10 PCR with Pfu]] -> Protocol: Thermal cycler program: [[Team:LMU-Munich/Notebook/Protocols/17 Special thermal cycler programs/Touch down|Touch down]] Mixture see 9-1-10, twice 4a and 4b <font color="#009933">Agarose gel electrophorese of PCR 4a, 4b, 7a, gel extracted 4a and 4b</font> -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]] - 25min, 150V [[image:9-2-10apo2.jpg|400px|gel 9-2-10-2]] from left to right: 4a*, 4a, 4b*, 4b, 7a Phusion, 7a Pfu, Ladder -> result: 4b, 4b*: right bands (~330bp) -remain: false bands/no band [[image:9-2-10apo3.jpg|400px|gel 9-2-10-3]] from left to right: ladder, 4 columns pathway, 4a gelextr., 4b gelextr. -> results: slight right bands for 4a and 4b, no "primer clouds" anymore. == 9-03-2010 == <font color="#009933">Overlapping PCR 5 with Pfu and Phusion</font> template: 4a, 4b Primer 7,10 Mixture with Pfu {| |- |PCR4a (~15ng) |1.2µl |- |PCR4b (~15ng) |2µl (1:10) |- |Pfu |0.5µl |- |Primer *2 |2.5µl *2 |- |10x buffer |5µl |- |dNTP Mix |1µl |- |DMSO |1,25µl |- |H₂O |34.05µl |- |sum |50µl |- |} -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/10 PCR with Pfu| 10 PCR with Pfu]] PCR program: standard PCR program for Pfu, Annealing temperature: 54°C PCR mixture with Phusion ::{| |- |PCR4a (~15ng) |1.2µl |- |PCR4b (~15ng) |2µl (1:10) |- |dNTPs |1 µl |- |Phusion |0.5 µl |- |5xbuffer |10 µl |- |DMSO |1,25µl |- |H₂O |29.05µl |- |sum |50µl |} -> Protocol:[[Team:LMU-Munich/Notebook/Protocols/15 PCR with Phusion| 15 PCR with Phusion]] PCR program: standard PCR program for Phusion, Annealing temperature: 58°C <font color="#009933">New PCR7a with Pfu</font> template: 190-6; Primer: 13,14 Mixture with Pfu {| |- |template (~2ng) |1µl |- |Pfu |0.5µl |- |Primer *2 |2.5µl *2 |- |10x buffer |5µl |- |dNTP Mix |1µl |- |DMSO |1,25µl |- |H₂O |36.25µl |- |sum |50µl |- |} -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/10 PCR with Pfu| 10 PCR with Pfu]] PCR program: standard PCR program for Pfu, Gradient: 54.4°C, 57.8°C, 61.4°C, 65.0°C -> results: -7a: only "primer cluods" -5 Pfu: no defined product (slurred?) -5 Phusion: "primer clouds" == 9-04-2010 == weekend == 9-05-2010 == weekend == 9-06-2010 == charges for sequencing {| class="wikitable" border="1" |- ! name ! 4a-7 ! 4a-8 ! 4b-9 ! 4b-10 ! 3-3 ! 3-6 ! 6-11 ! 6-12 |- | DNA | 4a; 2.4µl | 4a; 2.4µl | 4b; 0.5µl | 4b; 0.5µl | 3; 2µl | 3; 2µl | 6; 0.5µl | 6; 0.5µl |- | primer [1pmol/µl] | 7; 3.2µl | 8; 3.2µl | 9; 3.2µl | 10; 3.2µl | 3; 3.2µl | 6; 3.2µl | 11; 3.2µl | 12; 3.2µl |- | Tris (10mM); pH 8.2 | 1.4µl | 1.4µl | 3.3µl | 3.3µl | 1.8µl | 1.8µl | 3.3µl | 3.3µl |} every charge 7µl <font color="#009933">New overlapping PCR 5 with Pfu</font> template: 4a, 4b; 15ng Primer 7,10 Mixture {| |- |PCR4a (~7ng) |0.56µl |- |PCR4b (~8ng) |1.1µl (1:10) |- |Pfu |0.5µl |- |Primer *2 |2.5µl *2 |- |10x buffer |5µl |- |dNTP Mix |1µl |- |DMSO |1,25µl |- |H₂O |36.6µl |- |sum |50µl |- |} -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/10 PCR with Pfu| 10 PCR with Pfu]] PCR program: standard PCR program for Pfu, Annealing temperature: 46.1°C,48.5°C, 51.1°C -> results: "primer clouds" <font color="#009933">New PCR 7a with Pfu</font> new dilution of 190-6; 1:100 {| |template[2ng/µl] |1µl |2µl |- |pfu polymerase |0.5µl |0.5µl |- |primer13 |2.5µl |2.5µl |- |primer14 |2.5µl |2.5µl |- |buffer |5µl |5µl |- |dNTP Mix |1µl |1µl |- |DMSO |1,25µl |1.25µl |- |H₂O |36.25µl |35.25 |- |sum |50µl |50µl |- |} <font color="#009933">Agarose gel electrophoresis of 7a</font> -> results: "primer clouds" <font color="#009933">charges for sequencing</font> for PCR 1,7b and 9 {| class="wikitable" border="1" |- ! name ! 1-1 ! 1-2 ! 7b-15 ! 7b-16 ! 9-20 ! 9-21 ! 10-22 ! 10-23 |- | DNA | 1[1:10]; 1.74µl | 1[1:10]; 1.74µl | 7b; 3µl | 7b; 3µl | 9; 1.78µl | 9; 1.78µl | 10; 3.8µl | 10; 3.8µl |- | primer [1pmol/µl] | 1; 3.2µl | 2; 3.2µl | 15; 3.2µl | 16; 3.2µl | 20; 3.2µl | 21; 3.2µl | 22; 3.2µl | 23; 3.2µl |- | Tris (10mM); pH 8.2 | 2.06µl | 2.06µl | 0.8µl | 0.8µl | 2.02µl | 2.02µl | 0µl | 0µl |} every charge 7µl == 9-07-2010 == <font color="#009933">Agarose gel electrophorese of PCR gel extractions</font> 150V, 25min -> Protocol:[[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]] -result: [[image:7-9-10-1.jpg|400px|gel photo]] from left to right: PCR 1,2a,2b,3,4a,/,7b,9,10,/,ladder,pathway {| |- !PCR nr. !1 !2a !2b !3 !4a !7b !9 !10 |- |expected band (bp) |492 |332 |772 |1087 |330 |402 |808 |1888 |- |shown band(s) |550,200 |300 |750 |1100 |300 |450 |900,1500 |1900 |- |clean charge | |x |x |~x |x |x | |x |- |} <font color="#009933">new PCR for PCR6</font> PCR mixture for PCR6 ::{| |- |Concentration |Low |High |- |template |1 µl (1:100) |15µl (1:100) |- |primer (11,12) |2.5 µl*2 | |- |dNTPs |1 µl | |- |Pfu |0.5 µl | |- |10xbuffer |5 µl | |- |DMSO |1,25µl | |- |H₂O |36.25 µl |22.25 µl |- |sum |50µl | |} -> Protocol: Thermal cycler program: [[Team:LMU-Munich/Notebook/Protocols/17 Special thermal cycler programs/Touch down|Touch down]], 62°C-52°C, 30 cycles by 55°C == 9-08-2010 == <font color="#009933">Restriction digestion of eGFP, PCR6, ccdBamp</font> [[Image:Apo_8.9.10.jpg|thumb|right|Gel photo of (left to right) PCR6(5ng/100ng) template]] as prepatation for ligation -> Protocol [[Team:LMU-Munich/Notebook/Protocols/5_Restriction_digest|5 Restriction digestion]] {| !eGFP ! !SV40PA=PCR6 ! !ccdBamp ! |- |DNA |5µl=300ng |DNA |1µl=37,5ng |DNA |2µl=115ng |- |Buffer MC |2µl |Buffer D |2µl |Buffer H |2µl |- |BSA 1:10 |2µl |BSA 1:10 |2µl |BSA 1:10 |2µl |- |EcoRI |0,5µl |XbaI |0,5µl |EcoRI |0,5µl |- |SpeI |0,5µl |PstI |0,5µl |PstI |0,5µl |- |H₂O |13µl |H₂O |14µl |H₂O |13µl |- |sum |23µl |sum |20µl |sum |20µl |- |} 1:30h 37°C, 20min 80°C <font color="#009933">Agarose gel electrophoresis of PCR6</font> -> Protocol:[[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]] 25min, 150V <font color="#009933">Ligation 1</font> {| |eGFP |14,4µL |(144ng) |- |SV40PA |4,8µL |(48ng) |- |ccdBamp |2,9µL |(100ng) |- |T4 Buffer 10x |3µL | |- |T4 Ligase |0,5µL | |- |H₂O |4,4µL | |- |sum |30µL | |} Ligation at 22,5°C for 30 min, denaturation at 65°C for 10 min. Concentration of DNA in Ligation 1: {| |540ng |A260/A280=2,4 |} <font color="#009933">Transformation</font> -> Protocol [[Team:LMU-Munich/Notebook/Protocols/18 Competent cells2|18 competent cells2]] The incubation time for the cells is here 1 hour. == 9-09-2010 == <font color="#009933">Agarose gel electrophoresis of PCR 1, 3, 4a, 5, 7a, 9</font> Agarose gel electrophoresis of PCR 1, 3, 4a, 5, 7a, 9 -> protocol [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]] results: "primer-clouds", PCR 9: no band <font color="#009933">Plasmid extraction of ccdBcam, ccdBamp, Bak, CMV, PhiC31o</font> -> Protocol [[Team:LMU-Munich/Notebook/Protocols/4 Plasmid extraction from cells|4 Plasmid extraction from cells]] {| |Plasmid |concentration [ng/µL] |A260/A280 |- |ccdBcam |22.5 |1.0 |- |ccdBamp |42.5 |1.2 |- |Bak |17.5 |0.8 |- |CMV |10.0 |0.7 |- |PhiC310 |60 |1.4 |} Eluated with H₂O instead of the Eluation Buffer. <font color="#009933">New PCR 1, 3, 4a, 5, 7a, 9, 10 with phusion</font> {| class="wikitable" border="1" |- ! PCRnr. ! 1 ! 3 ! 4a ! 4b ! 5 ! 7a ! 9 ! 10 |- | template | pDS7 (1:100); 4µl | 2a; 0.9µl+2b; 0.6µl | Bak; 0.5µl | Bak; 0.5µl | 4a; 0.8µl+4b; 1µl | 190-6 (1:100); 1µl | eGFP (1:25); 2µl | PhiC31o (1:10); 1µl |- | primer [10pmol/µl] | 1,2; 2.5µl | 3,6; 2.5µl | 7,8; 2.5µl | 9,10; 2.5µl | 7,10; 2.5µl | 13,14; 2.5µl | 20,21; 2.5µl | 22,23; 2.5µl |- | H₂O | 28µl | 30.5µl | 31.5µl | 31.5µl | 30.2µl | 31µl | 30µl | 31µl |- |Annealing temperature |51.5°C |53.1°C |53.1°C |51.5°C |53.1°C |57.5°C |61°C |57.5°C |} + in each assay: dNTP mix: 1µl, 5x Phusion buffer: 10µl, DMSO:1.5µl, Phusion:0.5µl sum: 50µl Program: standard Phusion PCR, 29 cycles; annealing temperatures: see above -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/15 PCR with Phusion| 15 PCR with Phusion]] [[Image: apo 09.09 1.jpg|400px|gel electrophoresis of PCR 1, 3, 4a, 5, 7a, 9, 10]] [from left to right: PCR 1, 3, 4a, 5, 7a, 9, 10, Ladder] PCR3, 9, 10 with right bands. <font color="#009933">New PCR 1, 4a, 4b, 5, 7a with phusion</font> {| class="wikitable" border="1" |- ! PCRnr. ! 1 ! 4a ! 4b ! 5 ! 7a |- | template | pDS7 (1:10); 1µl | Bak 1 ; 1µl | Bak 1; 1µl | 4a, 4b; 2 x 1.5µl | pCT 190-6 (1:40); 1µl |- | primer | 1, 2; 2 x 2.5µl | 7, 8; 2 x 2.5µl | 9, 10; 2 x 2.5µl | 7, 10; 2 x 2.5µl | 13, 14; 2x 2.5µl |- | H₂O | 31µl | 31µl | 31µl | 30µl | 30µl |- | Annealing temperature | 52°C | 52°C | 48°C | 50°C | 55°C |} + each assay with dNTP-mix (1µl), 5xBuffer (10µl, DMSO (1.5µl), Phusion (0.5µl) -> sum: 50µl -> Protocol [[Team:LMU-Munich/Notebook/Protocols/17 Special thermal cycler programs/Touch down phusion|Touch down 59 with phusion]], 30 cycles with gradient appropriate for the annealing temperatures above. == 9-10-2010 == <font color="#009933">Agarose gel electrophoresis of PCR 1, 4a, 4b, 5, 7a</font> -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]] [[Image: 9-10-10.jpg|400px|gel electrophoresis of PCR 1, 4a, 4b, 5, 7a]] [From left to right: Ladder, 1, 4a, 4b, 5, 7a] Only 4a has been amplified successfully. <font color="#009933">Agarose gel extraction of PCR 3, 4a, 5, 9, 10</font> [[Image: apo-10-9-10-2.jpg|400px|gel electrophoresis of PCR 3, 4a, 5, 9, 10]] from left to right: PCR 3, band ~700bp, PCR 4a, band ~300bp, PCR 5, bands ~550bp (5*), ~650bp (5), PCR 9, band ~800bp, PCR 10, band ~1900bp results: {| class="wikitable" border="1" |- ! ! PCR 3 ! PCR 4a ! PCR 5 ! PCR 5* ! PCR 9 ! PCR 10 |- | concentration [ng/µl] | 20 | 10 | 5 | 30 | 35 | 25 |- | A260/A280 | 1.6 | 1.333 | 2.0 | 2.0 | 1.750 | 2.0 |} -> Protocol [[Team:LMU-Munich/Notebook/Protocols/12 Gel extraction or PCR Clean up| 12 Gel extraction or PCR Clean up (Promega kit)]] == 9-11-2010 == weekend == 9-12-2010 == weekend == 9-13-2010 == charges for sequencing (retry of 6.9.) {| class="wikitable" border="1" |- ! name ! 4a-7 ! 4a-8 ! 4b-9 ! 4b-10 ! 3-3 ! 3-6 ! 6-11 ! 6-12 |- | DNA | 4a; 2.4µl | 4a; 2.4µl | 4b; 0.5µl | 4b; 0.5µl | 3; 2µl | 3; 2µl | 6; 0.5µl | 6; 0.5µl |- | primer [1pmol/µl] | 7; 3.2µl | 8; 3.2µl | 9; 3.2µl | 10; 3.2µl | 3; 3.2µl | 6; 3.2µl | 11; 3.2µl | 12; 3.2µl |- | Tris (10mM); pH 8.2 | 1.4µl | 1.4µl | 3.3µl | 3.3µl | 1.8µl | 1.8µl | 3.3µl | 3.3µl |} every charge 7µl <font color="#009933">New PCR7a with Taq</font> template: p190-6 Primer 13,14 Mixture {| |- |template (~4ng) |2µl (1:100) |- |MasterMix for Taq |10µl |- |Primer *2 |1.5µl *2 |- |DMSO |0,5µl |- |H₂O |4.5µl |- |sum |20µl |- |} PCR program: touchdown PCR with Taq ::{| |1: 94°C 2' ---- |- |2: 94°C 30" |- |3: 64°C/62°C/60°C/58°C/56°C/54° 30" |- |4: 72°C 2' |- |5: (for each temperature)repeat 2-4 2x ---- |- |6: 94°C 30" |- |7: 58°C 30" |- |8: 72°C 2' |- |9: repeat 6-8 29x ---- |- |10: 72°C 10' |- |11: 15°C break |} -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/21 PCR with Taq Mastermix|21 PCR with Taq Mastermix]] <font color="#009933">overnight culture inoculated of</font> :CMV (amp) :ccdB (amp) :ccdB (cam) :pC31o (amp) :Bak (amp) <font color="#009933">New PCR7a with Phusion Hot Start</font> with 2ng and 4 ng template ::{| |- |template |2ng |4ng |- |H₂O |54µl |53µl |- |Buffer 5x |20µl |20µl |- |Primer (13,14) |2*10µl |2*10µl |- |DMSO |3µl |3µl |- |Hot Start |1µl |1µl |} total: 100µl each, divided into 5 charges program: ::98°C 30sec ::---- ::98°C 10sec ::gradient: 50°C, 53°C, 56,6°C, 60,2°C, 64,5°C 30sec 30 cycles ::72 15sec ::---- ::72°C 5min ::12°C forever -> Protocol:[[Team:LMU-Munich/Notebook/Protocols/20 PCR with Phusion Hot Start|20 PCR with Phusion Hot Start]] == 9-14-2010 == <font color="#009933">Agarose gelelectrophoresis of PCR 7a</font> -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]] bands 1 to 5: 2ng of template DNA bands 6 to 10: 4ng of template DNA -> no bands <font color="#009933">new PCR 7a and PCR 8 (without mutation)</font> with mastermix, without DMSO {| |- |number: |7a diluted |7a undiluted |8 |- |mastermix |50µl |50µl |10µl |- |primer |2*10µl |2*10µl |2*2µl |- |H₂O |29µl |29µl |5µl |- |template |1µl 190-6 (1:100) |1µl 190-6 |1µl 190-6 (1:100) |} 7a diluated and undiluated: divided into 3 charges 7a diluated: 1,2,3; 7a undiluated: 4,5,6 {| |- |1 |2 |3 |4 |5 |6 |8 |- |48°C |52°C |56,1°C |48°C |52°C |56,1°C |52°C |} program: ::{| |94°C 2' ---- |- |94°C 30" |- |48°C/52°C/56,1°C 30" |- |72°C 1,5' |- |(for each temperature)repeat 30x ---- |- |72°C 5' |} <font color="#009933">Plasmid extraction of ccdB (amp) and ccdB (cam)</font> -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/4 Plasmid extraction from cells|4 Plasmid extraction from cells]] results: ::ccdB(amp): 105ng/µl A260/280: 2,00 ::cddB(cam): 102ng/µl A260/280: 1,952 == 9-15-2010 == <font color="#009933">Agarose gelelectrophoresis of PCR 7a diluted &7a undiluted & "8"(without mutation)</font> -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]] [[Image:9 15 2010.jpg|400px|9 15 10 Gelphoto]] from left to right: 7a: 1:100 diluted: 48°C, 52°C, 56.1°C, undiluted: 48°C, 52°C, 56.1°C, 8 without Mutation weak right band (with fuzz)for 7a undiluated with best result for 56°C -> <font color="#009933">new PCR 7a and "8"</font> with mastermix Taq -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/21 PCR with Taq Mastermix|21 PCR with Taq Mastermix]] template: 190-6, 1:10 and 1:5 temp: 56°C and 58°C {| |- |number |7a |7a |"8" |"8" |- |Mastermix |20µl |20µl |20µl |20µl |- |H₂O |15µl |15µl |15µl |15µl |- |Primer |(13,14) 2*2µl |(13,14) 2*2µl |(13,16) 2*2µl |(13,16) 2*2µl |- |Template |190-6 (1:5) 1µl |190-6 (1:10) 1µl |190-6 (1:5) 1µl |190-6 (1:10) 1µl |} 2 charges each: one for 56°C and one for 58°C annealing temperature program: ::{| |94°C 2' ---- |- |94°C 30" |- |56°C/58°C 30" |- |72°C 1,5' |- |(for each temperature)repeat 30x ---- |- |72°C 5' |} <font color="#009933">Restriction Digestion of PCR 1 (17.8.), PCR 3 (10.9.), PCR 5₁ (10.9.), PCR 5₂ (10.9.), PCR 6 (17.8.), PCR 9 (10.9.), PCR 10 (30.8.) for ligation with vector</font> EcoR1 + Pst1 with Buffer H; 50ng DNA {| | |1 |3 |5₁ |5₂ |6 |9 |10 |- |H₂O |13µl |12,5µl |5µl |13µl |13,5µl |13,5µl |12,5µl |- |Buffer H |2µl |2µl |2µl |2µl |2µl |2µl |2µl |- |BSA (1:10) |2µl |2µl |2µl |2µl |2µl |2µl |2µl |- |DNA |2µl |2,5µl |10µl |2µl |1,5µl |1,5µl |2,5µl |- |EcoR1 |0,5µl |0,5µl |0,5µl |0,5µl |0,5µl |0,5µl |0,5µl |- |Pst1 |0,5µl |0,5µl |0,5µl |0,5µl |0,5µl |0,5µl |0,5µl |} -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/5 Restriction digest|5 Restriction digestion]] <font color="#009933">Agarose gelelectrophoresis of PCR 7a 1-4 & "8" 1-4(without mutation)</font> -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]] -> bad results <font color="#009933">purification of restriction digestion</font> {| |- | |concentration (ng/µl) |A₂₆₀/A₂₈₀ |(supposed) lenght |- |1 |20 |1,000 |492 |- |3 |5 |1,000 |1087 |- |5₁ |22,5 |1,8 |688 |- |5₂ |7,5 |1,5 |688 |- |6 |10 |1,333 |237 |- |9 |20 |1,6 |808 |- |10 |17,5 |1,75 |1888 |} -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/12 Gel extraction or PCR Clean up| 12 Gel extraction or PCR Clean up (Promega kit)]] <font color="#009933">Ligation with pSB1C3 (2072bp)</font> {| |- |for |# |backbone |insert (µl) |charge (µl) |Buffer 10x (µl) |H₂O (µl) |- |100ng |1 |4 |7,12 |20 |2 |6,38 |- |50ng |3 |2 |31,48 |40 |4 |2,02 |- |100ng |5₁ |4 |8,85 |20 |2 |4,65 |- |100ng |5₂ |4 |26,56 |40 |4 |4,94 |- |100ng |6 |4 |6,86 |20 |2 |13,36 |- |100ng |9 |4 |11,7 |20 |2 |1,8 |- |100ng |10 |4 |31,24 |40 |4 |4,26 |} plus 1 µl T4 ligase in each charge -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/22 Ligation|22 Ligation]] <font color="#009933">new PCR 7a & "8"</font> with mastermix Taq 56°C {| |- | |7a-1 |7a-2 |"8"-1 |"8"-2 |- |mastermix (µl) |10 |10 |10 |10 |- |template |190-6 1µl |190-6 2µl |190-6(1:10) 1µl |190-6(1:10) 2µl |- |primer |13&14 2*1µl |13&14 2*1µl |13&16 2*1µl |13&16 2*1µl |- |H₂O |7µl |6µl |7µl |6µl |} sum: 20µl each pcr-program: ::{| |94°C 2' ---- |- |94°C 30" |- |56°C 30" |- |72°C 1,5' |- |(for each temperature)repeat 30x ---- |- |72°C 5' |- |12°C forever |} -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/21 PCR with Taq Mastermix|21 PCR with Taq Mastermix]] == 9-16-2010 == <font color="#009933">Agarose gelelectrophoresis of PCR 7a-1, PCR 7a-2, PCR 8-1, 8-2 and of all three PCR 6 we ever made (in order to control whether we have extracted the right fragment (237) or just the primerdimers)</font> -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]] [[Image:9 16 10.jpg|400px|9 16 10 Gelphoto]] From left to right: 6₂, 6₁, 6, Ladder, 7a-1, 7a-2, 8-2, 8-1 ::results: ::6-1 and 6-2: ? ::6: ok ::7a-1,7a-2,"8"-1,"8"-2: primerdimer-problem -> we ordered new primers! <font color="#009933">transformation of the ligations 1,3,5₁, 5₂, 6, 9, 10</font> -> Protocol:[[Team:LMU-Munich/Notebook/Protocols/3 Transformation|3 Transformation]] ->plated on plates with chloramphenicol <font color="#009933">again PCR 1,3,5,6,9,10 in order to increase the profit (Ausbeute) for ligation</font> {| |- | |1 |3 |5 |6 |9 |10 |- |template |prev TRE(1:30) 1µl |2a(1:5) 1µl + 2b 0,5µl |4a 0,5µl + 4b 0,5µl |SV40PA (pcDNA3)(1:50) 1µl |eGFP(1:5) 1µl |PhiC310(1:6) 1µl |- |primer |1&2 2*2,5µl |3&6 2*2,5µl |7&10 2*2,5µl |11&12 2*2,5µl |20&21 2*2,5µl |22&23 2*2,5µl |- |T_m |54°C |50°C |49°C |56°C |58°C |58°C |- |mastermix (µl) |25 |25 |25 |25 |25 |25 |- |DMSO |1,25µl |1,25µl |1,25µl |1,25µl |1,25µl |1,25µl |- |H₂O (µl) |17,75 |17,25 |17,75 |17,75 |17,75 |17,75 |} pcr-program: ::{| |94°C 2' ---- |- |94°C 30" |- |T_m (see above) 30" |- |72°C 1,5' |- |(for each temperature)repeat 30x ---- |- |72°C 5' |} -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/21 PCR with Taq Mastermix|21 PCR with Taq Mastermix]] == 9-17-2010 == <font color="#009933">Analysis of the transformation from yesterday</font> Colonies on plates when 100µl or pellet plated: {| |- | |1 |3 |5 |6 |9 |10 |- |pellet |no |yes |yes |yes |yes |yes |yes |- |100µl |no |yes |yes |yes |yes |yes |yes |} <font color="#009933">again: transformation of ligation 1</font> ->Protocol: [[Team:LMU-Munich/Notebook/Protocols/3 Transformation|3 Transformation]] <font color="#009933">Agarose gelelectrophoresis of yesterday's PCR 1,3,5,6,9,10(without mutation)</font> -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]] [[Image:9 17 10apo.jpg| 400px |9 17 10 Gelphoto]] From left to right: PCR1, PCR3, PCR5, Ladder, PCR6, PCR9, PCR10 -> right band for PCR 3,5,6,9,10; no band for PCR 1 [[Image:9 17 10apo2.jpg| 400px |9 17 10 Gelphoto2]] From left to right: Ladder, PCR3, PCR5, PCR9, PCR10 Bands took: 3₂: Band short over 1000bp, 3₁: Band short under 1000bp (probably 3₂ right), 5 the highest band, 9 upper band, 10 upper band <font color="#009933">new PCR 1,5,6</font> {| |- | |1 |5 |6 |- |template |pTRE Rev 0,5µl |4a 1,5µl + 4b 1,5µl |pcDNA3 0,5µl |- |primer |1&2 2*2,5µl |7&10 2*2,5µl |11&12 2*2,5µl |- |buffer 10x |5µl |5µl |5µl |- |dNTPs |1µl |1µl |1µl |- |Pfu |0,5µl |0,5µl |0,5µl |- |H₂O |26,75 µl |24,25 µl |26,75 µl |- |T<sub<m</sub> |51°C |50°C |55°C |} -> Protocol: pcr-program: standard Pfu [[Team:LMU-Munich/Notebook/Protocols/10 PCR with Pfu| 10 PCR with Pfu]] <font color="#009933">Restriction digestion of PCR products, ccdB Plasmids and Biobricks for the 3A Method</font> {| |Name |H₂O |Buffer (each 2µl) |BSA (1:10) |DNA Volume |DNA Mass |Enzyms (2*0.5µl) |- |ccdBa |25 µl |H |2µl |20µl |~600ng |E+P |- |ccdBa |25µl |H |2µl |20µl |~700ng |E+P |- |1 |43µl |MC |2µl |2µl |~500ng |E+S |- |3 |25µl |B |2µl |20µl |~200ng |X+P |- |Primer 18+19 |25µl |B |2µl |10µl+10µl |? |X+P |- |5₁ |25µl |MC |2µl |20µl |~100ng |E+S |- |5₂ |25µl |MC |2µl |20µl |~600ng |E+S |- |6 |35µl |B |2µl |10µl |~400ng |X+P |- |9 |25µl |MC |2µl |20µl |~700ng |E+S |- |10 |25µl |MC |2µl |20µl |~400ng |E+S |- |eGFP |35µl |MC |2µl |10µl |~550ng |E+S |- |CMV1 |35µl |H |2µl |10µl |~530ng |E+P |} -> Protocol:[[Team:LMU-Munich/Notebook/Protocols/5 Restriction digest|5 Restriction digestion]] <font color="#009933">Colony PCR of Ligations 3, 5₁, 5₂, 6, 9, 10 and PCR of CMV1 (to test if right)</font> 10 1-4, 9 1-4, 6 1-4, 5₁ 1-4, 5₂ 1-4, 3 1-4 Colonies pickt and put in following Mix: {| |PCR Mastermix |10µl |- |Biobrick Primer F |1.5µl |- |Biobrick Primer R |1.5µl |- |H₂O |7µl |} CMV1: {| |PCR Mastermix |10µl |- |Biobrick Primer F |1.5µl |- |Biobrick Primer R |1.5µl |- |Template |2µl |- |H₂O |5µl |} -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/21 PCR with Taq Mastermix|21 PCR with Taq Mastermix]] <font color="#009933">Agarose Gel electrophoresis of 3, 5₁, 5₂, 6, 9, 10 and CMV1 (to test if right)</font> -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]] [[Image:17 9 10apo.jpg|400px|17 9 10 Gelphoto]] Above: From left to right: 3.1, 3.2, 3.3, 3.4, 5₁.1, 5₁.2, 5₁.3, 5₁.4, Ladder, 5₂.1, 5₂.2, 5₂.3, 5₂.4 Below: From left to right: 6.1, 6.2, 6.3, 6.4, 9.1, 9.2, 9.3, 9.4, Ladder, 10.1, 10.2, 10.3, 10.4, CMV1 -> right bands for 6.3 and 6.4, CMV1 ~1200bp (we think that this is right, as Biobrick sequenzing information indicates that it isn't 654bp but about 1200bp) <font color="#009933">PCR clean up of PCR Gel extraction 3₁, 3₂, 5, 9, 10, PCR Product 6 and digestion PCR1, Primer 18+19, R5₁, R5₂, PCR6, PCR9, PCR10, PCR3</font> -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/12 Gel extraction or PCR Clean up| 12 Gel extraction or PCR Clean up (Promega kit)]] == 9-18-2010 == weekend == 9-19-2010 == weekend == 9-20-2010 == <font color="#009933">Dephosphorylation of linearized ccdB amp and ccdB cam</font> Add 1µl TSAP to digested vectors. Incutation: 37°C 15min; Inhibition: 74°C 15min <font color="#009933">Ligation and 3A-Assemblies</font> Jump-or-Die Ligations: 1a, 1b (5₁),1b (5₂), 2, 3 Cut'N'Survive Ligations: 1a, 2b, Biobricks for both Systems: CMV, PCR1 (tet-on-promoter) Mixtures: - 3A Assemblies (everything except CMV, PCR1): Inserts: each 8µl; ccdB vector: 1µl; T4 Ligase: 0.5µl; T4 Ligase Buffer: 2µl; H₂O: 1µl - Biobricks: Insert: 10µl; ccdB vector: 1µl; T4 Ligase: 0.5µl; T4 Ligase Buffer: 2µl; H₂O: 7µl Incubation: 2:30h 16°C; Inhibition: 10 min 65°C ->Protocols: [[Team:LMU-Munich/Notebook/Protocols/22 Ligation|22 Ligation]], [[Team:LMU-Munich/Notebook/Protocols/13 3A Method for Biobrick assembly| 13 3A Method for Biobrick assembly]] <font color="#009933">Colony PCR of PCR product ligations with pSB1C3</font> Mixture: PCR Mastermix:130µl ; PrimerF:19,5µl ; PrimerR:19,5µl ; H₂O:91µl -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/21 PCR with Taq Mastermix|21 PCR with Taq Mastermix]] <font color="#009933">Agarose Gel Electrophoresis of Colony PCRs</font> -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]] [[Image:20.9.10.jpg|400px|Colony PCRs]] above from left to right: pathway/ladder/3.5/3.6/3.7/3.8/5₁.5/5₁.6/5₁.7/5₁.8/5₂.5/5₂.6/5₂.7/5₂.8 below from left to right: ladder/6.5/6.6/6.7/6.8/9.5/9.6/9.7/9.8/10.5/10.6/10.7/10.8 == 9-21-2010 == <font color="#009933">Agarose Gel Electrophoresis of PCRs 1, 5, 6, and Colony PCR 3.8, 5₁.7, 5₂.6, 9.7, 10.5, 10.6</font> -> Bad results, probably too much cells in PCR mix -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]] <font color="#009933">Colony PCR of 3.8, 5₁.7, 5₂.6, 9.7, 10.5, 10.6 again and of the 3A Ligations of yesterday as well as the Ligation of PCR1 and CMV with pSB1C3</font> PCR Master Mix: 60µl Primer F: 9µl Primer R: 9µl H₂O: 42µl ->22charges à 5µl -> Protocol:[[Team:LMU-Munich/Notebook/Protocols/21 PCR with Taq Mastermix|21 PCR with Taq Mastermix]] <font color="#009933">PCR 7a with new Primers</font> {| |- |template 190-6 (1:10) |1µl |- |Master Mix |25µl |- |Primer 13k (short) |3,75µl |- |Primer 14 |3,75µl |- |H₂O |16,5µl |} sum: 50µl same with Primer 13l (long) ->Protocol: [[Team:LMU-Munich/Notebook/Protocols/21 PCR with Taq Mastermix|21 PCR with Taq Mastermix]] <font color="#009933">Agarose Gel Electrophoresis of Colony PCRs and PCR 7a</font> different ladder: [[image:fermentas ladder mix.jpg|thumb|right|fermentas dna ladder mix (source:http://www.fermentas.com/en/products/all/dna-electrophoresis/generuler-dna-ladders/sm0333-generuler-mix]] [[image:21 9 10apo2.jpg|400px|gelfoto 21 9 10 apo2]] key: JD=Jump-or-Die; CS=Cut'N'Survive; first number=Ligation number (see schedule); second number=colonie number (marked on the plate) from left to right:7ak(short primer), 7al(long primer), JD.1a.1, JD.1a.2,JD.1b5₁.1, JD.1b5₂.2, JD.1b5₂.1, JD.1b5₂.2, JD.2.1, JD.2.2, JD.3₁.1, JD.3₁.2, CS.1a.1, CS.1a.2 verified products: 7a (~800bp); JD.2.1; JD.2.2(each ~1000bp);JD.3₁.1(~2000bp); CS.1a.2(~1500bp) no bands shown: CS.2b.1, CS.2b.2, PCR1.1, PCR1.CMV,3.8, 5₁.7, 5₂.6, 9.7, 10.5, 10.6 -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]] == 9-22-2010 == <font color="#009933">Inoculate Colonies</font> inoculated in 4ml LB-medium with appropriate antibiotic <font color="#009933">Plated residual transformated E. colis (9-20-10) (where we had few colonies on plates) </font> <font color="#009933">Colony PCRs</font> for 22*5µl charges: {| |PCR Mastermix |55µl |- |Primer F |8,25µl |- |Primer R |8,25µl |- |H₂O |38,5µl |- |sum |110µl |} -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/21 PCR with Taq Mastermix|21 PCR with Taq Mastermix]] Annealing temperature: 53°C <font color="#009933">PCR Purification of 7a</font> charges 7ak and 7al had not been labeled, so they were renamed. results: 7a₁: 135ng/µl; A260/A280=1.7 7a₂: 133ng/µl; A260/A280=1.7 <font color="#009933">PCR8 with Phusion Hot Start II</font> -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/20 PCR with Phusion Hot Start|20 PCR with Phusion Hot Start]] {| |- |template 7a 1:100 |3µl ~4ng (850bp) |- |template 7b 1:10 |2µl ~2ng (402bp) |- |Buffer 5X |10µl |- |Phusion |0.5µl |- |H₂O |27µl |- |Primer 13 |2.5µl |- |Primer 16 |2.55µl |- |DMSO) |1.5µl |- |dNTP mix |1µl |} Program: annealing temperature: 63°C, elongation time: 40sec <font color="#009933">Agarose gel electrophoresis of Colony PCRs and PCR8</font> new ladder: fermentas FastRuler (TM) Middle Range DNA Ladder http://www.fermentas.com/en/products/all/dna-electrophoresis/fastruler-dna-ladders/sm111-fastruler-middle [[image: 9 22 10 beschriftet.jpg|400px|gelfoto]] some results: PCR 8 (lanes 23, 24): no band Colony PCRs CS.2b.10, PCR1cam.3, PCR1cam.4, CMVcam.4, CMVcam.4, JD.1b5₁.10 (lanes 7,8,9,10,11,14): show right bands <font color="#009933">Plasmid extraction of "over-day" cultures ligation-7(=BB4),-8(=BB4),-9(=BB5),-12(=BB9) and pSB1C3</font> ->Protocol: [[Team:LMU-Munich/Notebook/Protocols/4 Plasmid extraction from cells|4 Plasmid extraction from cells]] concentrations (ng/µl - A_260/280): ::BB4(7):57,5 - 1,643 ::BB4(8):15 - 1,5 ::BB5(9):173 - 1,816 ::BB7(12):55,0 - 1,571 ::pSB1C3: 12,5 - 1,250 <font color="#009933">new PCR 7b in order to get better results</font> {| |- |Master Mix |25µl |- |H₂O |16,5µl |- |Primer 15 |3,75µl |- |Primer 16 |3,75µl |- |template 190-6 (1:10) |1µl |} <font color="#009933">overnight-cultures</font> inoculated of: CS2bK10=BB11; PCP1K3+4=BB1; CMV-C-K3+4; JD1b5₁K10=BB2; pSB1C3 == 9-23-2010 == <font color="#009933">Restriction digestion of BB5 for the 3A Method</font> {| |Name |H₂O |Buffer D |BSA (1:10) |DNA Volume |DNA Mass |Enzyms (2*0.5µl) |- |BB5 |11 µl |2µl |2µl |4µl |~500ng |X+P |- |} sum: 20µl -> Protocol:[[Team:LMU-Munich/Notebook/Protocols/5 Restriction digest|5 Restriction digestion]] <font color="#009933">Plasmid extraction of overnight cultures BB2, BB11, CMV in cam and pSB1C3</font> ->Protocol: [[Team:LMU-Munich/Notebook/Protocols/4 Plasmid extraction from cells|4 Plasmid extraction from cells]] concentrations (ng/µl - A_260/280): ::BB2:232 - 1,603 ::BB11:170 - 1,889 ::CMV in cam:272 - 1,703 ::pSB1C3: 290 - 1,758 The pSB1C3 colony was pink (produced a red pigment or something like that, so we make a new transformation in order to extract only pSB1C3 and not some other vectors. <font color="#009933">Agarose gelelectrophoresis of PCR 7b and pathway restriction digestions</font> -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]] [[Image:23 9 10 1.jpg|thumb|right|Gelphoto]] From left to right: PCR7b, pathway (3x),ladder (5000,2000,850,400,100bp) result: PCR 7b shows right band with ~400bp <font color="#009933">PCR clean up of PCR 7b</font> -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/12 Gel extraction or PCR Clean up| 12 Gel extraction or PCR Clean up (Promega kit)]] result: 90ng/µl; A260/A280= 1,895 <font color="#009933">Restriction digestion of BB2 and BB11</font> {| |Name |H₂O |Buffer D |BSA (1:10) |DNA Volume |DNA Mass |Enzyms (2*0.5µl) |- |BB2 |10 µl |2µl |2µl |5µl |~1.2mg |X+P |- |} sum: 20µl {| |Name |H₂O |Buffer D |BSA (1:10) |DNA Volume |DNA Mass |Enzyms (2*0.5µl) |- |BB11 |7 µl |2µl |2µl |8µl |~1.3mg |X+P |- |} sum: 20µl -> Protocol [[Team:LMU-Munich/Notebook/Protocols/5 Restriction digest|5 Restriction digestion]] <font color="#009933">Ligation of BB1 and BB6</font> -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/22 Ligation|22 Ligation]] {| |Name |H₂O |T4 Ligase-Buffer |PCR1 |Primer 18+19 |ccdBAmp |T4 Ligase |- |BB1 |9.5 µl |2µl |5µl |2µl |1µl |0.5µl |- |} {| |Name |H₂O |T4 Ligase-Buffer |CMV E+S |BB5 |ccdBCam |T4 Ligase |- |BB6 |6.5 µl |2µl |8µl |2µl |1µl |0.5µl |- |} <font color="#009933">PCR 8 with 7b(new) plus 7a-2 with Phusion Hot Start II; Primer 16 plus 13k and 13l</font> {| |- |Buffer 5x |10µl |- |H₂O |27µl |- |dNTPs |1µl |- |Primer 16 |2,5µl |- |Primer 13k short |2,5µl |- |DMSO |1,5µl |- |template 7a(1:100)(850bp) |3µl -> 4ng |- |template 7b(new)(1:100)(402bp) |2µl -> 2ng |- |Phusion Hot Start II |0,5µl |} :->same with Primer 13l long<- <font color="#009933">Overnight culture of PCR1 in cam- Vector</font> new inoculation of PCR1 in cam, Colony 3 in 5ml LB+cam [[Image:23 9 10 2apo.jpg|thumb|right|From left to right: PCR8k,PCR8l,ladder (5000,2000,850,400,100bp) ]] <font color="#009933">Transformation of pSB1C3, BB1, BB6</font> New transformation of pSB1C3 from Spring distribution because the old colony was pink. Transformation of BB1 and BB6. BB1 plated on amp-Agar, BB6 and pSB1C3 plated on cam-Agar -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/19 Transformation SSS|19 Transformation (Sina Science Services)]] <font color="#009933">Agarose gelelectrophoresis of PCR8 (long and short primer 13)</font> PCR8k=short primer 13, PCR8l=long primer 13 -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/11 Agarose gel electrophoresis| 11 Agarose gel electrophoresis]] result: PCR 8k and 8l shows right band with ~1200bp <font color="#009933">PCR purification of PCR 8</font> -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/12 Gel extraction or PCR Clean up| 12 Gel extraction or PCR Clean up (Promega kit)]] result: ~70ng/µl; A260/A280=1.75 == 9-24-2010 == <font color="#009933">New "over-day" culture of pSB1C3</font> Inoculate one culture from the ligation yesterday in 4ml LB+cam <font color="#009933">Ligation of BB7₂</font> Mixture: Inserts: each 5µl; ccdB vector: 1µl; T4 Ligase: 0.5µl; T4 Ligase Buffer: 2µl; H₂O: 7µl Incubation: 5h 18°C; Inhibition: 20 min 65°C ->Protocols: [[Team:LMU-Munich/Notebook/Protocols/22 Ligation|22 Ligation]], [[Team:LMU-Munich/Notebook/Protocols/13 3A Method for Biobrick assembly| 13 3A Method for Biobrick assembly]] <font color="#009933">Colony PCRs of BB1 and BB6</font> 2 Colonys were picked from each plate -> 4 charges Mixture for 4 charges with 5µl: {| |PCR Mastermix 2x |10µl |- |Primer F |1.5µl |- |Primer R |1.5µl |- |H₂O |7µl |- |sum |20µl |- |} + Colony [[Image: apo-24.9.10.jpg|200px|thumb|right|from left to right: 1₁, 1₂, 6₁ and 6₂]] -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/21 PCR with Taq Mastermix|21 PCR with Taq Mastermix]] Annealing temperature: 53°C, standard protocol <font color="#009933">Plasmid extraction of new PCR1 in cam vector</font> -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/4 Plasmid extraction from cells|4 Plasmid extraction from cells]] results: {| | concentration | 82.5ng/µl |- | A260/A280 | 1.737 |} <font color="#009933">Agarose Gelelektrophoresis of 1₁, 1₂, 6₁ and 6₂</font> <font color="#009933">Plasmid extraction of pSB1C3 in cam vector</font> -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/4 Plasmid extraction from cells|4 Plasmid extraction from cells]] results: {| | concentration | 22.5ng/µl |- | A260/A280 | 1.5 |} == 9-25-2010 == weekend == 9-26-2010 == weekend == 9-27-2010 == <font color="#009933">Colony PCR of BB7₂, BB6 and BB1 plus a negativ charge as reference</font> {| | PCR Mastermix | 17.5µl |- | Primer forward | 2.625µl |- | Primer revers | 2.7µl |- | H₂O | 12.1µl |- | sum | 35µl = 5µl per charge |} -> Protocol [[Team:LMU-Munich/Notebook/Protocols/24 Colony PCR|24 Colony PCR]] <font color="#009933">Ligation of BB10</font> {| | CMV© E+S | 5µl |- | PCR 8l X+P | 5µl |- | ccdBamp | 1µl |- | T4 Ligase-Buffer | 2µl |- | T4 Ligase | 0.5µl |- | H₂O | 6.5µl |- | sum | 20µl |} -> Protocol [[Team:LMU-Munich/Notebook/Protocols/22 Ligation|22 Ligation]] <font color="#009933">Ligation of BB001(PCR1 in ©), BB017(PCR5 in ©), BB018(PCR6 in ©) and BB021(PCR9 in ©)</font> {| | ccdB | 1µl |- | BB001/017/018/021 | 5µl |- | T4 Ligase-Buffer | 2µl |- | T4 Ligase | 0.5µl |- | H₂O | 11.5µl |- | sum | 20µl |} -> Protocol [[Team:LMU-Munich/Notebook/Protocols/22 Ligation|22 Ligation]] <font color="#009933">Restriction digestion of PCR3₂, 8l, 10 and Primer 18+19 with E+P</font> {| class="wikitable" border="1" |- ! ! template ! H₂O ! BSA [1:10] ! Buffer H ! EcoRI ! PstI |- | PCR3₂ | 10µl | 5µl | 2µl | 2µl | 0.5µl | 0.5µl |- | PCR8l | 10µl | 5µl | 2µl | 2µl | 0.5µl | 0.5µl |- | Primer 18+19 | 2x7.5µl | -- |2µl | 2µl | 0.5µl | 0.5µl |- | PCR10 | 10µl | 5µl | 2µl | 2µl | 0.5µl | 0.5µl |} -> Protocol [[Team:LMU-Munich/Notebook/Protocols/5 Restriction digest|5 Restriction digestion]] == 9-28-2010 == <font color="#009933">Plating BB017, BB021, BB018, BB001, BB7₂, BB10</font> {| class="wikitable" border="1" |- | Amp | BB7₂ | BB10 | | |- | Cam | BB001 | BB017 | BB018 | BB021 |} <font color="#009933">Colony PCR BB1, BB6, BB7₂ with DreamTaq (each 2 colonies)</font> -> Protocol: [[Team:LMU-Munich/Notebook/Protocols/16 PCR with DreamTaq| 16 PCR with DreamTaq]] PCR mixture for PCR7a ::{| |- |template |pick up colony from plate |- |primers*2 |0.25µl*2 |- |dNTPs |0.5µl |- |DreamTaq |0.05µl |- |10xbuffer |0.5µl |- |H₂O |3.45µl |- |sum |5µl |} Primers for colony PCR: BBseqF, BBseqR Annealing temperature: 53°C <font color="#009933">Ligation of BB2, BB019, BB020, BB016, BB022</font> {| class="wikitable" border="1" |- ! ! BB2 ! BB019 ! BB020 ! BB016 ! BB022 ! Volume |- | PCR | 5₂ | 8 | | 3₂ | 10 | each 5µl |- | PCR | 6 | | | | | each 5µl |- | Primer | | | 18+19 | | | each 5µl |- | ccdBamp | 1µl | | | | | |- | ccdBcam | | 1µl | 1µl | 1µl | 1µl | |- | T4 Ligase-Buffer | 2µl | 2µl | 2µl | 2µl | 2µl | |- | T4 Ligase | 0.5µl | 0.5µl | 0.5µl | 0.5µl | 0.5µl | |- | H₂O | 6.5µl | 11.4µl | 11.4µl | 11.4µl | 11.4µl | |- | sum | 20µl | 20µl | 20µl | 20µl | 20µl | |} == 9-29-2010 == plates BB7₂, BB017, BB10, BB021, BB001, BB018: nothing grown -> thrown into trash <font color="#009933">colony-PCR of BB2/8, BB019, BB016, BB020, BB022 with Taq</font> ->protocol: [[Team:LMU-Munich/Notebook/Protocols/25 PCR with Taq|PCR with Taq]] {| |- |H₂O |6,7µl |- |Buffer 10x |1µl |- |MgCl₂ |1µl |- |dNTPs |0,2µl |- |Primer forw. seq f |0,5µl |- |Primer rev. seq r |0,5µl |- |Taq |0,1µl |} template: pick colony sum: 10µl; we made 100µl ->10 charges (2 colonies of each plate) tube numbers: {| class="wikitable" border="1" |- |1 |2 |3 |4 |5 |6 |7 |8 |9 |10 |- |BB2/BB8 - 1 |BB2/BB8 - 2 |BB016 - 1 |BB016 - 2 |BB019 - 1 |BB019 - 2 |BB020 - 1 |BB020 - 2 |BB022 - 1 |BB022 - 2 |} PCR-program: taq53 agarose-gelelektrophoresis -> no bands visible <font color="#009933">new plates made of BB1, BB4, BB6, BB7</font> <font color="#009933">Ligations BB7₂, BB017 (mit PCR5₁), BB10, BB021, BB001, BB018</font> {| class="wikitable" border="1" |- | |PCR (5 µl each) |PCR (5µl each) |ccdB(amp) |ccdB© |Buffer |T4 Ligase |H₂O |- |BB7₂O |1 |3₂ |1µl | |2µl |0,5µl |6,5µl |- |BB017 |5₁ | | |1µl |2µl |0,5µl |11,5µl |- |BB10 |CMV© |PCR8 |1µl | |2µl |0,5µl |6,5µl |- |BB021 |9 | | |1µl |2µl |0,5µl |11,5µl |- |BB001 |1 | | |1µl |2µl |0,5µl |11,5µl |- |BB018 |6 | | |1µl |2µl |0,5µl |11,5µl |} <font color="#009933">restriction degestions</font> of PCR6, PCR5₂, PCR3₂, PCR1, ccdB(amp), ccdB(cam) {| class="wikitable" border="1" |- |template |digestion with |volume |- |PCR 6 |Xba1 & Pst1 |I. 10µl II. 3µl |- |PCR5₂ |EcoR1 & Pst1 |20µl |- |PCR3₂ |Xba1 & Pst1 |10µl |- |PCR1 |EcoR1 & Spe1 |2µl |- |ccdB(amp) |EcoR1 & Pst1 |20µl |- |cddB(cam) |EcoR1 & Pst1 |20µl |} ->protocol: [[Team:LMU-Munich/Notebook/Protocols/5 Restriction digest|5 Restriction digestion]] <font color="#009933">colony-PCR of BB4/c3, BB6/c7, BB7₂/c5, BB1/c5, (BB11 as control) with Taq</font> ->protocol: [[Team:LMU-Munich/Notebook/Protocols/25 PCR with Taq|PCR with Taq]] Mastermix: {| |- |H₂O |32µl |- |Buffer 10x |5µl |- |MgCl₂ |5µl |- |dNTPs |1µl |- |Primer forw. seq f |2,5µl |- |Primer rev. seq r |2,5µl |- |Taq |0,5µl |- |DMSO |1,5µl |- |sum |50µl |- |} ->10µl for each charge template: pick colony; BB11: 2ng tube numbers: {| class="wikitable" border="1" |- |1 |2 |3 |4 |5 |- |BB4-c3 |BB6-c7 |BB7₂-c5 |BB1-c5 |BB11 |- |} PCR program:T_A = 53°C; elongation time=1min == 9-30-2010 == <font color="#009933">Colony PCR of BB2/BB8, BB6 K7&K8 and test PCR of BB11 with new dilutions and primers</font> {| | Colony/BB11 | 2.5µl |- | H₂O | 32µl |- | dNTP | 1µl |- | Primer F | 2.5µl |- | Primer R | 2.5µl |- | MgCl₂ | 5µl |- | DMSO | 1.5µl |- | Buffer 10x (with KCl) | 5µl |- | Taq Polymerase | 0.5µl |- | sum | 56µl |} -> Protocol [[Team:LMU-Munich/Notebook/Protocols/24 Colony PCR|24 Colony PCR]] <font color="#009933">PCR purification of the digestions of 6I, 6II, 5₂, 3₂, 1</font> {| | | concentration [ng/µl] | A260/A280 |- | 6II | 32.5 | 1.444 |- | ccdBamp | 0.0 | --- |- | ccdBcam | 5.0 | 1.000 |- | 6I | 0.0 | --- |- | 5₂ | 20.0 | 1.143 |- | 3₂ | 27.5 | 1.57 |- | 1 | 25 | 1.667 |} -> Protocol [[Team:LMU-Munich/Notebook/Protocols/12 Gel extraction or PCR Clean up| 12 Gel extraction or PCR Clean up (Promega kit)]] <font color="#009933">Transformation of BB018, BB7₂, BB001, BB017, BB10 and BB021</font> -> Protocol [[Team:LMU-Munich/Notebook/Protocols/19 Transformation SSS|19 Transformation SSS]] <font color="#009933">Plasmidextraction of the following overnight cultures: BB020(K1), BB018(K5), BB6(K5), BB022(K1), BB016(K1), BB019(K1), BB2(K1) and BB018(K3)</font> {| | | concentration [ng/µl] | A260/A280 |- | BB020(K1) | 163 | 1.711 |- | BB018(K5) | 92.5 | 1.85 |- | BB6(K5) | 120 | 1.778 |- | BB022(K1) | 105 | 1.75 |- | BB016(K1) | 85 | 1.889 |- | BB019(K1) | 105 | 1.909 |- | BB2(K1) | 190 | 1.81 |- | BB018 | 110 | 1.833 |} -> Protocol [[Team:LMU-Munich/Notebook/Protocols/4 Plasmid extraction from cells|4 Plasmid extraction from cells]] == 10-01-2010 == <font color="#009933">new test PCR of BB5 and BB11 with 20µl and 50µl volume</font> Master Mix {| | H₂O | 67µl |- | dNTP | 2µl |- | Primer F | 5µl |- | Primer R | 5µl |- | MgCl₂ | 10µl |- | Buffer 10x (with KCl) | 10µl |- | Taq Polymerase | 1µl |- | sum | 100µl |} template: BB5/50µl: 5ng, 20µl: 1.5ng; BB11/20µl: 3ng ->protocol: [[Team:LMU-Munich/Notebook/Protocols/25 PCR with Taq|PCR with Taq]] [[Image:01.10.10. apo.jpg|01.10.10. Gel photo]] from left to right: ladder, BB5/50µl, BB5/20µl, BB11/20µl. BB5/50µl and BB11/20µl show the right band. == 10-02-2010 == weekend == 10-03-2010 == weekend == 10-04-2010 == sequencing cycle, clean and run {| class="wikitable" border="1" |- ! ! CMV (c) ! PCR 1 (c) ! BB4 ₇ ! BB4 ₈ ! BB018K5 ! BB018K3 ! BB6K5 ! BB2K1 |- | template | 1µl | 2µl | 3µl | 3,8µl | 2µl | 2µl | 2µl | 1µl |- | primer (1:100) | 3,2 µl | 3,2 µl | 3,2 µl | 3,2 µl | 3,2 µl | 3,2 µl | 3,2 µl | 3,2 µl |- | 10mM TrispH 8,5 | 2,8 µl | 1,8 µl | 0,8 µl | 0 µl | 1,8 µl | 1,8 µl | 1,8 µl | 1,8 µl |- |name of tube | 028 FIR | 001 F/R | 4-7 F/R | 4-8 F/R | 018-5 F/R | 018-3 F/R | 6-5 F/R | 2-1 F/R |} PCR {| class="wikitable" border="1" |- ! ! 019 ! 022 ! 016 ! 020 |- | template (1:100 = 2ng) | 2µl | 2µl | 2,5µl | 1,5µl |- | Primer F | 1µl | 1µl | 1µl | 1µl |- | Primer R | 1µl | 1µl | 1µl | 1µl |- | dNTPs | 0,4µl | 0,4µl | 0,4µl | 0,4µl |- | Taq | 0,2µl | 0,2µl | 0,2µl | 0,2µl |- | Buffer | 2µl | 2µl | 2µl | 2µl |- |MgCl₂ | 2µl | 2µl | 2µl | 2µl |- |H₂O | 11,4µl | 11,4µl | 10,9µl | 11, |- |colour of tube | orange | rose | green | blue |} == 10-05-2010 == <font color="#009933">Colony PCRs of BB6, BB4, BB021, BB022, CMV©, BB001, BB016, BB1, BB2_5₂, BB7₂, BB017, BB018, BB019, BB020</font> <font color="#009933">agarose gel electrophoresis</font> schedule of the agarose gel electrophoresis: {| class="wikitable" border="1" | gel 1 | BB6K9 | BB6K10 | BB6K11 | BBK12 | BB6K13 | BB4K5 | ladder | BB4K6 | BB4K7 |- | | BB021K14 | BB021K13 | BB021K12 | BB021K11 | BB022K18 | BB022K19 | ladder | BB022K17 | BB022K16 |- | gel 2 | BB022K12 | BB022K11 | CMV©K4 | CMV©K5 | BB001K5 | BB001K6 | BB001K7 | BB001K8 | ladder |- | | BB016K17 | BB1K16 | BB1K15 | BB1K8 | BB1K7 | BB2_5₂K1 | BB2_5₂K2 | BB2_5₂K3 | ladder |- | gel 3 | BB017K14 | BB017K13 | BB017K12 | BB017K11 | BB018K17 | BB018K16 | BB018K15 | BB018K14 | ladder |- | | BB019K6 | BB019K5 | BB019K4 | ladder | BB019K3 | BB020K7 | BB020K8 | BB020K6 | BB020K5 |} {| class="wikitable" border="1" | gel 1 | BB4K8 | BB4K9 | BB4K10 | BB021K10 | BB021K15 | | | | |- | | BB022K15 | BB022K14 | BB022K13 | --- | --- | | | | |- | gel 2 | BB001K9 | BB001K10 | BB016K11 | BB016K12 | BB016K13 | BB016K14 | BB016K15 | BB016K16 | --- |- | | --- | BB2_5₂K4 | BB2_5₂K5 | BB7₂K12 | BB7₂K11 | BB7₂K10 | BB7₂K9 | BB017K16 | BB017K15 |- |gel 3 | BB018K13 | BB018K12 | BB018K11 | BB018K10 | BB018K9 | | | | |- | | BB020K4 | BB020K3 | --- | --- | | | | | |} Gelbilder und Auswertung einfügen [[Image:5.10.10apo1,2,3.jpg|400px|9 15 10 Gelphoto]] <font color="#009933">overnight cultures of BB6K3, BB4K10, CMV©K4, BB001K5, BB001K9, BB016K11, BB1K8, BB2_5₂K3, BB7₂, BB017K14, BB018K10, BB019K6</font> == 10-06-2010 == <font color="#009933">Plasmidextraction of the following overnight cultures: BB6(K13), BB4(K13), CMVc (K4), BB001(K5), BB001(K9), BB016(K11), BB1(K8), BB2(K3), BB7(K10), BB017(K14), BB018(K10) and BB019(K6) </font> {| | | concentration [ng/µl] | A260/A280 |- | BB6(K13) | 265 | 1.8 |- | BB4(K10) | 40 | 2.0 |- | CMVc(K4) | 97.5 | 1.8 |- | BB001(K5) | 30 | 1.7 |- | BB001(K9) | 17.5 | 1.7 |- | BB016(K11) | 45 | 1.7 |- | BB1(K8) | 57.5 | 1.9 |- | BB2(K3) | 60 | 1.8 |- | BB7(K10) | 30 | 1.7 |- | BB017(K14) | 12.5 | 1.5 |- | BB018(K10) | 35 | 1.8 |- |BB019(K6) | 37.5 | 1.5 |- |} -> Protocol [[Team:LMU-Munich/Notebook/Protocols/4 Plasmid extraction from cells|4 Plasmid extraction from cells]] == 10-07-2010 == <font color="#009933"> colony PCR of BB021, BB022 with Taq (53°C) </font> {| | Primer fw | 0.5 µl |- | Primer rev | 0.5 µl |- | dNTPs | 0.2 µl |- | Taq | 0.1 µl |- | 10xbuffer | 1 µl |- | MgCl₂ | 1 µl |- | H₂O | 6.7 µl |- |} == 10-08-2010 == <font color="#009933"> plasmide extractions from 10-06-2010 were put in a lyophilizer and redissolved in less water (10-20 µl) </font> {| | | concentration [ng/µl] | A260/A280 |- | BB4(K10) | 258 | 1,981 |- | BB001(K5) | 325 | 1.94 |- | BB001(K9) | 205 | 1.907 |- | BB7(K10) | 170 | 1.744 |- | BB017(K14) | 215 | 1.955 |- | BB018(K10) | 500 | 1.88 |- |BB019(K6) | 293 | 1.95 |- |} == 10-09-2010 == <font color="#009933">Plasmid extraction BB018K5, BB018K6, BB021K17 and BB021K24</font color> concentration of these samples to be measured on Monday -> Protocol [[Team:LMU-Munich/Notebook/Protocols/4 Plasmid extraction from cells|4 Plasmid extraction from cells]] == 10-10-2010 == weekend == 10-11-2010 == <font color="#009933">Plasmid extraction BB018K5, BB018K6, BB021K24 and BB021K17 from 10-09-2010 </font> {| | | concentration [ng/µl] | A260/A280 |- | BB018K5 | 55 | 1.571 |- | BB018K6 | 92,5 | 1.762 |- | BB021K24 | 27.5 | 1.833 |- | BB021K17 | 37.5 | 1.508 |- |} <font color="#009933">Colony PCR BB01, BB018, BB6, BB252</font> {| | stripe 1 | BB001K5 | K6 | K7 | K8 | K9 | K9 | K10 | BB252 K2 |- | stripe 2 | BB252 K3 | K4 | K5 | BB6 K9 | K10 | K11 | K12 | K13 |- | stripe 3 | BB018 K9 | K10 | K11 | K12 | K13 | K14 | K15 | K16 |- |single sample |BB018 K17 |- |} gel picture colony PCR foto einfügen! <font color="#009933">Sequencing 8,9 and 10 (cycle, clean and run)</font> {| class="wikitable" border="1" |- ! ! BB001K9 for ! BB001K9 rev ! BB4K16 for ! BB4K16 rev ! BB001K5 for ! BB001K5 rev ! BB2_5₂K3 ! BB2_5₂K3 |- | template | 1µl | 1µl | 1µl | 1µl | 0,5µl | 0,5µl | 3,8µl | 3,8µl |- | primer (1:100) | 3,2 µl | 3,2 µl | 3,2 µl | 3,2 µl | 3,2 µl | 3,2 µl | 3,2 µl | 3,2 µl |- | 10mM TrispH 8,5 | 2,8 µl | 2,8 µl | 2,8 µl | 2,8 µl | 3,3 µl | 3,3 µl | 0 µl | 0 µl |- |name of tube | 31-F | 31-R | 44-F | 44-R | 24-F | 24-R | 32-F | 32-R |} {| class="wikitable" border="1" |- ! ! BB7₂K10 for ! BB7₂K10 rev ! BB017K14 for ! BB017K14 rev ! BB018K10 for ! BB018K10 rev ! BB019K6 for ! BB019K6 rev |- | template | 1,5µl | 1,5µl | 1µl | 1µl | 0,5µl | 0,5µl | 0,8µl | 0,8µl |- | primer (1:100) | 3,2 µl | 3,2 µl | 3,2 µl | 3,2 µl | 3,2 µl | 3,2 µl | 3,2 µl | 3,2 µl |- | 10mM TrispH 8,5 | 2,3 µl | 2,3 µl | 2,8 µl | 2,8 µl | 3,3 µl | 3,3 µl | 3 µl | 3 µl |- |name of tube | 14-F | 14-R | 21-F | 21-R | 13-F | 13-R | 34-F | 34-R |} {| class="wikitable" border="1" |- ! ! CMV for ! CMV rev ! BB018K5 for ! BB018K5 rev ! BB018K6 for ! BB018K6 rev ! BB021K17 ! BB021K17 |- | template | 2µl | 2µl | 3,8µl | 3,8µl | 2µl | 2µl | 3,8µl | 3,8µl |- | primer (1:100) | 3,2 µl | 3,2 µl | 3,2 µl | 3,2 µl | 3,2 µl | 3,2 µl | 3,2 µl | 3,2 µl |- | 10mM TrispH 8,5 | 1,8 µl | 1,8 µl | 0 µl | 0 µl | 1,8 µl | 1,8 µl | 0 µl | 0 µl |- |name of tube | CMV-F | CMV-R | 185-F | 185-R | 186-F | 186-R | 2117-F | 2117-R |} <font color="#009933">ligation BB020 BB022, Bb6 and BB5₂</font color> -> Protocol [[Team:LMU-Munich/Notebook/Protocols/19 Transformation SSS|19 Transformation SSS]] {| class="wikitable" border="1" |- ! ! BB020 ! BB022 ! BB6 ! BB5₂ |- | insert (5µl) | 18+19 | PCR 10 | CMV | PCR5<sub<2</sub> |- | insert (5µl) | - | - | BB5 | PCR6 |- | ccdB (1µl) | c | c | c | a |- | buffer | 2µl | 2µl | 2µl | 2µl |- | T4-ligase | 0,5µl | 0,5µl | 0,5µl | 0,5µl |- | H₂O | 11,5µl | 11,5µl | 6,5µl | 6,5µl |} <font color="#009933">PCR to test self-made taq</font color> {| | template eGFP | 1µl |- | Primer 20,21 | each 1µl |- | buffer (10x) | 2µl |- | MgCl | 2µl |- | dNTPs | 0,2µl |- | taq | 0,3µl to 0,7µl (0,1µl steps) |- | H₂O | fill to 20 µl |- |} anealing temperature 58°C, elongation time 30'' == 10-12-2010 == == 10-13-2010 == text == 10-14-2010 == text == 10-15-2010 == text == 10-16-2010 == weekend == 10-17-2010 == weekend == 10-18-2010 == text == 10-19-2010 == text == 10-20-2010 == text == 10-27-2010 == text == 10-28-2010 == text == 10-29-2010 == text == 10-30-2010 == weekend {{:Team:LMU-Munich/Templates/Page Footer}}

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