|
|
| Line 108: |
Line 108: |
| | By red eye screening, we identified the strain 13 and strain 14 to be the successfully transformed lines. Photographs for the transformed red (orange) eye fly (strain 13) were taken. | | By red eye screening, we identified the strain 13 and strain 14 to be the successfully transformed lines. Photographs for the transformed red (orange) eye fly (strain 13) were taken. |
| | <br><br> | | <br><br> |
| - | Drosophila S2 cells (2.5 X 10<sup>5</sup> cells per well) were plated on the 24 well plate and cultured in the Schneider’s Drosophila medium containing 10% fetal bovine serum at 25 ℃.
| + | |
| - | <br><br>
| + | |
| | | | |
| | <h2>September 20th</h2> | | <h2>September 20th</h2> |
| | <br> | | <br> |
| - |
| |
| | By red eye screening, we identified the strain 23 and strain 29 to be the successfully transformed lines. | | By red eye screening, we identified the strain 23 and strain 29 to be the successfully transformed lines. |
| - | <br><br>
| |
| - | At 20 min after removing the medium, 25 uL of serum free medium containing the 1 uL of siLentfect and 200 ng each of pAct5C-GAL4 DNA and pUAS-EGFP DNA was added to the well. As a negative control, 25 uL of serum free medium containing the 1 uL of siLentfect and 200 ng of pUAS-EGFP DNA alone was added to the well. Similarly, 25 uL of serum free medium containing the 1 uL of siLentfect and 200 ng each of pAct5C-GAL4 DNA and pUAS-LacZ DNA was added to the well. As a negative control for this experiment, 25 uL of serum free medium containing the 1 uL of siLentfect and 200 ng of pUAS-LacZ DNA alone was added to the other well. At 4 hours after transfection, the transfection medium was removed and the Schneider’s Drosophila medium containing 10% fetal bovine serum was added to each well. The cells were incubated for 48 or 72 hours at 25 ℃.
| |
| | <br><br> | | <br><br> |
| | | | |
| Line 129: |
Line 125: |
| | <h2>September 22nd</h2> | | <h2>September 22nd</h2> |
| | <br> | | <br> |
| - |
| |
| | Red eye screening was continued. | | Red eye screening was continued. |
| - | <br><br>
| |
| - | The cells transfected with pAct5C-GAL4 DNA and pUAS-EGFP DNA were examined under the confocal lazer microscope (Olympus, Fv10i).
| |
| - | <br>
| |
| - | Results: 12.6% cells were observed to be EGFP-positive, while only o.5% cells were apparently EGFP-positive for the negative control cells, indicating that the P-element plasmid, pUAS-EGFP DNA, Biobrick part (BBa_K758006) is truly functional in Drosophila cells. We therefore decided to send this prasmid to iGEM Headquarters.
| |
| - | <br><br>
| |
| - | Leftpanel: cells co-transfected with pAct5C-GAL4 DNA and pUAS-EGFP DNA
| |
| - | <br>
| |
| - | Right panel: cells transfected with pUAS-EGFP DNA alone (negative control)
| |
| - | <br><br>
| |
| - | <img src="https://static.igem.org/mediawiki/2012/b/b5/実験ノート１.png" width="282" height="282"> <img src="https://static.igem.org/mediawiki/2012/c/cd/実験ノート２.png" width="282" height="282">
| |
| - | <br><br>
| |
| - | The cells transfected with pAct5C-GAL4 DNA and pUAS-LacZ DNA were fixed with cold methanol for 20 min or 4% paraformaldehyde for 15 min. After fixation, cells were washed with 0.5% Triton X-100 in PBS, then masked with 5% normal goat serum in PBS for 1 hour. Then the monoclonal anti-beta galactosidase antibody (X500 dilution in PBS containing 5% normal goat serum) was added to the fixed cells and incubated for 16 hours at 4℃.
| |
| | <br><br> | | <br><br> |
| | + | |
| | | | |
| | | | |
| Line 149: |
Line 133: |
| | <h2>September 23rd</h2> | | <h2>September 23rd</h2> |
| | <BR> | | <BR> |
| - |
| |
| | By red eye screening, we identified the strain 56 and strain 65 to be the successfully transformed lines. These transformed flies are kept at 25℃ and later will be further inspected for chromosome linkage of the transgene. | | By red eye screening, we identified the strain 56 and strain 65 to be the successfully transformed lines. These transformed flies are kept at 25℃ and later will be further inspected for chromosome linkage of the transgene. |
| - | <br><br>
| |
| - | After incubation with the first antibody, the cells were washed with PBS (three times for 10 min each). Then the Alexa-conjugated anti-mouse IgG (X400 dilution in PBS containing 5% normal goat serum) added to the cells and incubate for 4 hours at 4℃. After washing with PBS (three times for 10 min each), cells were mounted with mounting medium in DAPI and examined under the confocal lazer microscope (Olympus, Fv10i).
| |
| - | Results: 10.1% cells co-transfected with pAct5C-GAL4 DNA and pUAS-LacZ DNA were observed to be LacZ-positive, while no LacZ-positive cell was detected for the negative control cells, indicating that the P-element plasmid, pUAS-LacZ DNA, Biobrick part (BBa_K758005) is truly functional in Drosophila cells. We therefore decided to send this plasmid to iGEM Headquarters.
| |
| - | <br><br>
| |
| - | After 72 hours incubation, the cells transfected with pAct5C-GAL4 DNA and pUAS-EGFP DNA were examined under the confocal lazer microscope (Olympus, Fv10i). Results were nearly the same as those after 48 hours incubation.
| |
| - | <br><br>
| |
| - | Leftpanel: cells co-transfected with pAct5C-GAL4 DNA and pUAS-LacZ DNA
| |
| - | <br>
| |
| - | Right panel: cells transfected with pUAS-LacZ DNA (negative control)
| |
| - | <br><br>
| |
| - | <img src="https://static.igem.org/mediawiki/2012/a/a8/実験ノート３.png" width="282" height="282"> <img src="https://static.igem.org/mediawiki/2012/1/17/実験ノート４.png" width="282" height="282">
| |
| | <br><br> | | <br><br> |
| | | | |
"
