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| Line 814: |
Line 814: |
| | <li>skim-milk-plate assay kerUS and KerA - spread on prepared skim-milk agar plates (supernatant and cells) - 4 days incubation, room temperature (link results)</li></ul><br><br> | | <li>skim-milk-plate assay kerUS and KerA - spread on prepared skim-milk agar plates (supernatant and cells) - 4 days incubation, room temperature (link results)</li></ul><br><br> |
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| − | <h3>Preparation of chemically competent DH5alpha E. coli cells</h3>
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| − | <br>
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| − | Material:
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| − | <br>
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| − | <ul><li>LB media</li>
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| − | <li>TSS buffer </li>
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| − | <li>DH5alpha E. coli cells (o/n colonies on agar plates)</li></ul>
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| − | <br>
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| − | Method:
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| − | <br>
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| − | <ol><li>Pick one colony of the plate and transfer into 5 mL of LB media. Grow the culture over night for 16-18 hours at 37 deg. celsius. </li>
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| − | <li>Transfer 1 mL of the overnight culture into a shaking flask with 99 mL of LB media. Measure optical density (OD) at 600 nm and incubate culture at 37 deg. celsius (shaking) to an OD of 0,5. </li>
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| − | <li>Divide the 100 mL into two 50 mL tubes and incubate 10 min on ice. </li>
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| − | <li>Spin the tubes at 3000 rpm for 10 minutes at 4 deg. celsius. </li>
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| − | <li>Resuspend the pellet of competent cells with 10 % TSS buffer (5 mL). </li>
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| − | <li>Aliquot 100 µL of the cell solution into 1.5 mL microtubes (all steps on ice!). </li>
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| − | <li>Store the competent cells at -80 deg. celsius.</li> </ol>
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| − | <br>
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| − | <h3>Heat-shock Transformation of E. coli cells</h3>
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| − | <br>
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| − | Material:
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| − | <br>
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| − | <ul><li>SOC media </li>
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| − | <li>Agar plates with appropriate antibiotic</li></ul>
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| − | <br>
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| − | Method:
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| − | <br>
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| − | <ol><li>Thaw chemically competent cells on ice. </li>
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| − | <li>Transfer 50 µL of the cells into a 1.5 mL microtube, add 1 µL of the desired DNA and incubate on ice for 30 minutes. </li>
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| − | <li>Place the tube in a 42 deg. celsius water bath for 60 seconds. </li>
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| − | <li>After heat shock leave cells on ice for 5 minutes. </li>
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| − | <li>Add 950 µL of SOC media and shake cells for 2 hours at 37 deg. celsius. </li>
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| − | <li>Pipet 100 µL of the cells onto an appropriate plate and spread them using sterile glass beads. Incubate overnight at 37 deg. Celsius and hope for colonies in the morning to prove a successful transformation. </li></ol>
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| − | <br>
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| − |
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| − | <h3>Preparation of LB media</h3>
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| − | <br>
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| − | Material:
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| − | <br>
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| − | <ul><li>Tryptone</li>
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| − | <li>NaCl</li>
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| − | <li>Yeast extract</li></ul>
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| − | <br>
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| − | Method:
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| − | <br>
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| − | <ol><li>Fill a container (bottle) to about 60/70 % its volume with destilled water. </li>
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| − | <li>Add 10 g/L Tryptone, 10 g/L NaCl and 5 g/L of yeast extract. </li>
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| − | <li>Stir properly and fill up the remaining volume with distilled water. </li>
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| − | <li>Treat LB media by autoclave. </li></ol>
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| − | <br>
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Notebook
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Notebook - important experiments
26.07.2017
- transformation of kerUS (BBa_K1498000)
- preparation of antibiotic stock solutions: chloramphenicol (35 mg/mL) and ampicillin (100 mg/mL)
27.07.2017
- growing of a single colony (kerUS (BBa_K1498000)) in 5 mL LB media chloramphenicol (35 µg/mL] at 37 °C
- single colonies are plated on agar plates and incubated at 37 °C over night
28.07.2017
- Preparation of: miniprep (Jena Biosciences Kit)and
glycerol stock (kerUS (BBa_K1498000))
03.08.2017
- transformation of BBa_K1149002 and BBa_K1149003
04.08.17
- single colonies (BBa_K1149002 and BBa_K1149003) are plated on agar plates and incubated at 37 °C over night
09.08.17
- growing of a single colony (BBa_K1149002 and BBa_K1149003) in 5 mL LB media + chloramphenicol (35 µg/mL] at 37 °C
10.08.17
- Preparation of miniprep (Jena Biosciences Kit) and
glycerol stock (storage: -80 °C) (BBa_K1149002 and BBa_K1149003)
- SOC media preparation
- transformation pet19-LipB
21.08.2017
- preparation: preliminary test of esterase assay with EstCS2: Bba_K1149002 - preparation of o/n cultures (37°C)
22.08.2017
- preparation: preliminary test of esterase assay with EstCS2: Bba_K1149002 - glycerol stocks and induction with arabinose
23.08.2017
- preliminary test of esterase assay with EstCS2: Bba_K1149002 (link results)
28.08.2017
- preparation of electro-competent cells
31.08.2017
- transformation/electroporation with iGEM competent cells test kit DNA (o/n incubation, 37°C - transformation failed)
01.09.2017
- transformation/electroporation with pUC19 plasmid (o/n incubation, 37°C - transformation failed)
06.09.2017-08.09.17
- enzyme activity assay (cell pellet and supernatant) with BBa_K1149002 and pet19-LipB
12.09.2017
- preparation of chemo-competent cells
13.09.2017
- transformation with iGEM competent cells test kit DNA and pUC19 plasmid (o/n incubation, 37°C) - transformation successful
14.09.2017
- calculation - efficiency of the chemo-competent cells/pUC19:
efficiency E =(56cfu/0.001ng)*1000ng/(µL)=5.6*((10)^7 )cfu/µL
18.09.2017
- preparation InterLab study - transformation of 8 parts into DH5alph E. coli cells
19.09.17 – 21.09.17
- InterLab study LUDOX measurement
- enzyme activity assay with BBa_K1149002 of the supernatant - measurement in biological triplicates
- InterLab study Fluorescein measurement
22.09.2017
- InterLab study sample measurement (link results)
27.09.17 - 29.09.17
- enzyme activity assay with pet19-LipB of the supernatant - measurement in biological triplicates
06.10.2017
- Collaboration iGEM team Heidelberg: preparation mutagenesis plasmid activity assay - preparation of media, antibiotic-stocks and sugar-stocks + agar plates with different antibiotics
10.10.2017
- Collaboration iGEM team Heidelberg: preparation of different cultures + induction of the cells with arabinose (o/n incubation, 37°C)
11.10.2017
- Collaboration iGEM team Heidelberg: spread the o/n cultures on prepared agar plates (o/n incubation, 37°C)
16.10.2017
- preparation semi-quantitative keratinase-assay: spread kerUS, KerA and KerP on chloramphenicol/kanamycin agar plates (o/n incubation, 37°C)
- Preparation skim-milk-plate assay: preparation of skim-milk-agar-plates with chloramphenicol/kanamycin
18.10.2017
- M9 media preparation
- preparation semi-quantitative keratinase-assay: preparation of o/n cultures (37°C)
- Preparation skim-milk-plate assay: preparation of o/n cultures (37°C)
19.10.2017
- PCR - amplification of LipB - cloning of LipB in the iGEM standard plasmid (BBa_K1149002 is used)
- PCR - amplification of BBa_K1149002 (without EstCS2)
- semi-quantitative keratinase-assay: add 0.005 g of dried hair (65 °C, 1h) to o/n cultures (KerUS and KerA are induced with 1mM IPTG before) - 5 days incubation, 37°C (link results)
- skim-milk-plate assay kerP - spread on prepared skim-milk agar plates (supernatant and cells) - 4 days incubation, room temperature (link results)
20.10.2017
- SDS page, Gibson Assembly, Transformation (Zymos) - cloning of LipB in the iGEM standard plasmid (BBa_K1149002 is used)
- skim-milk-plate assay kerUS and KerA - spread on prepared skim-milk agar plates (supernatant and cells) - 4 days incubation, room temperature (link results)
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