Team:Munich/purify1.html
T7 DNA Extraction by Phenol Chloroform Precipitation
2018/05/17| Participants: | Keno Eilers |
| Protocol: | Phage T7 DNA Purification, Phenol Chloroform precipitation |
| Notes: | Still too much DNA from E. coli |
| Results: | DNA concentration: 55,5 ng/µL |
Cleanup of T7 DNA from 15.5.18 with NEB PCR Cleanup Kit
2018/05/19| Participants: | Nils O'Brien |
| Protocol: | NEB PCR Cleanup Kit, Agarose Gel electrophoresis |
| Notes: | Warming of columns on 40 °C for a few minutes before elution |
| Results: | No results, not possible with genome larger than 25kb |
Second Try: Cleanup of T7 DNA from 15.5.18 with NEB PCR Cleanup Kit
2018/05/20| Participants: | Nils O'Brien |
| Protocol: | NEB PCR Cleanup Kit, Agarose Gel electrophoresis |
| Notes: | Warming of columns on 50 °C for a few minutes before elution |
| Results: | No visible bands on 0,3 Agarose gel, did not work out for large fragments. End of experiments with this method |
T7 DNA Clean-Up T7 DNA
2018/05/22| Participants: | Keno Eilers |
| Protocol: | Phenol Chloroform precipitation |
Generating MS2 RNA Genome
2018/05/23| Participants: | Keno Eilers |
| Protocol: | Phenol Chloroform precipitation, cDNA Synthesis |
| Notes: | 1 µl 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 Eilers |
| Protocol: | Phenol Chloroform precipitation, cDNA Synthesis |
| Notes: | 1 µl DNAse I (NEB, 2.000 U/mL) added, incubate for 10 minutes at 37 °C |
| Results: | RNA concentration: 34900 ng/µl |
Optimization of DNA Precipitation out of T7 Phage
2018/05/23 - 2018/05/25| Participants: | Keno Eilers, Sophie Kurzbach, Nils O'Brien |
| Protocol: | Phage T7 DNA Purification, Agarose Gelelectrophoresis |
| 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 |
T7 DNA Isolation with Different Infection Times
2018/05/29| Participants: | Sophie Kurzbach |
| Protocol: | Phage T7 DNA Purification, Phenol Chloroform precipitation |
| Notes: | Tested transfection times: 12 min (before supernatant), 22 min (after supernatant), 32 min (cell lysis) |
| Results: | 12 min: 209 ng/µL, 22 min: 628 ng/µL, 32 min: 334 ng/µL |
T7 DNA Isolation 10 min and 32 min after Super Transfection + RNAse
2018/06/05 -2018/06/06| Participants: | Sophie Kurzbach |
| Protocol: | Phage T7 DNA Purification, Phenol Chloroform precipitation, PEG percipitant |
| Notes: | Better results than DNA without RNAse treatment, still too much E. coli DNA |
| Results: | DNA concentration of 10 min: 633 ng/µL DNA concentration of 32 min: 200 ng/µL |
T7 DNA Extraction by Phenol Chloroform Precipitation
2018/06/16-2018/06/18| Participants: | Keno Eilers |
| Protocol: | Phage T7 DNA Purification, Phenol Chloroform precipitation |
| Notes: | No notes |
| Results: | Good DNA: 10,37 nM |
DNA Purification of T7 DNA with Qiagen Genomic Tips 100/G
2018/06/22| Participants: | Brigit Tunaj |
| Protocol: | Qiagen Genomic Tip Kit |
| Notes: | Preparation of buffers as descripted on their homepage |
| Results: | Did not work out |
DNA Purification of T7 DNA with Qiagen Genomic Tips 100/G
2018/06/25| Participants: | Brigit Tunaj |
| Protocol: | Qiagen Genomic Tip Kit |
| Notes: | Increased DNA concentration 50 ug |
| Results: | Did not work out. Method not suitable for phage DNA. |