Difference between revisions of "Team:Munich/purify1.html"

 
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       <td> Still too much from E. coli
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       <td> Still too much DNA from E. coli
 
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    <figcaption class="figure-caption">0,3% Agarose gel, 2 log ladder </figcaption>
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    <figcaption class="figure-caption">0,3% Agarose gel, 2 log ladder, after 10 min infection</figcaption>
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<h4>T7 DNA Extraction by Phenol Chloroform Precipitation </h4>
 
<h4>T7 DNA Extraction by Phenol Chloroform Precipitation </h4>

Latest revision as of 02:22, 18 October 2018

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.