Team:Munich/purify1.html
T7 DNA extraction by phenol chloroform precipitation
2018/05/17| Participants: | Keno |
| Protocol: | T7 DNA Purification, phenol-chloroform precipitation |
| Notes: | still too much from E. coli |
| Results: | DNA concentration: 55,5 ng/uL |
Cleanup of T7 DNA from 15.5.18 with NEB PCR Cleanup Kit
2018/05/19| Participants: | Nils |
| Protocol: | NEB PCR-cleanup Kit, agarose gel |
| Notes: | warming of columns on 40 °C for a few minutes before elution |
| Results: | no results, not possible with genome larger than 25kb |
Contents
second try: Cleanup of T7 DNA from 15.5.18 with NEB PCR Cleanup Kit
2018/05/20
| Participants: | Nils |
| Protocol: | NEB PCR-cleanup Kit, agarose gel |
| Notes: | warming of columns on 50 °C for a few minutes before elution |
| Results: | no results, did not work out for large fragments |
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T7 DNA clean up T7 DNA
2018/05/22
| Participants: | Keno, Kilian |
| Protocol: | phenol chloroform precipitation |
| Results: | no results à testing PEG cutoff tomorrow |
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Generating MS2 RNA genome
2018/05/23| Participants: | Keno |
| Protocol: | cDNA from PCR product, phenol chloroform precipitation |
| Notes: | 1 ul DNAse I (NEB, 2.000 U/mL) added, incubate for 10 minutes at 37 °C |
| Results: | RNA concentration: 34900 ng/µl |
Generating MS2 RNA genome
2018/05/23
| Participants: | Keno |
| Protocol: | cDNA from PCR product, phenol chloroform precipitation |
| Notes: | 1 ul DNAse I (NEB, 2.000 U/mL) added, incubate for 10 minutes at 37 °C |
| Results: | RNA concentration: 34900 ng/µl |
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Generating MS2 RNA genome
2018/05/23
| Participants: | Keno |
| Protocol: | cDNA from PCR product, phenol chloroform precipitation |
| Notes: | 1 ul DNAse I (NEB, 2.000 U/mL) added, incubate for 10 minutes at 37 °C |
| Results: | RNA concentration: 34900 ng/µl |
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Optimization of DNA precipitation out of T7 Phage
2018/05/23 - 2018/05/25
| Participants: | Keno, Sophie K., Nils |
| Protocol: | T7 DNA purification, agarose gel, |
| Notes: | Step 11 of "T7 DNA purification" protocol: Optimization of PEG (63%) precipitation
Incubation for 42 minutes, then proceeded with centrifugation |
| Results: | 150 µl PEG: 302 ng/µl, 200 µl PEG: 164 ng/µl, 250 µl PEG: 278 ng/µl, 300 µl PEG: 113 ng/µl, 350 µl PEG: 279 ng/µl, 400 µl PEG: 216 ng/µl, 450 µl PEG: 530 ng/µl, 500 µl PEG: 422 ng/µl |
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T7 DNA Isolation with different infection times
2018/05/29| Participants: | Sophie K. |
| Protocol: | T7 DNA purification, phenol chloroform precipitant |
| Notes: | tested transfection times: 12 min (before supernatant), 22 min (after supernatant), 32 min (cell lysis) |
| Results: | 12 min: 209 ng/uL, 22 min: 628 ng/uL, 32 min: 334 ng/uL |
T7 DNA Isolation 10 min and 32 min after super transfection + RNAse
2018/06/05 -2018/06/06| Participants: | Sophie K. |
| Protocol: | T7 DNA purification, PEG precipitant, phenol chloroform precipitant |
| Notes: | better results than DNA without RNAse treatment, still too much E. coli DNA |
| Results: | DNA concentration of 10 min: 633 ng/uL DNA concentration of 32 min: 200 ng/uL |
T7 DNA extraction by phenol chloroform precipitation
2018/06/16-2018/06/18| Participants: | Keno |
| Protocol: | T7 DNA purification, phenol chloroform precipitant |
| Notes: | - |
| Results: | good DNA: 10,37 nM |