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EPF-Lausanne/16 July 2008
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Perhaps the protocol which we were using was not the right one, so we modify it according to the iGEM parts registry guidelines (2 hours incubation after the heatshock). | Perhaps the protocol which we were using was not the right one, so we modify it according to the iGEM parts registry guidelines (2 hours incubation after the heatshock). | ||
| - | Make competent cells with the Top10 grown before (2 solutions). One solution was grown at room temperature on the bench at rest. The other was left shaking at 20°C. Both were grown 16+ hours. OD of 0.375 and around 0.650 were deemed OK (still in the growing phase). They were processed and stocked according to the exact protocol from openwetware. | + | Make competent cells with the Top10 grown before (2 solutions). We divided each solution in two to make to batches(easier for the centrifugation stages). One solution was grown at room temperature on the bench at rest( - and ?). The other was left shaking at 20°C(A and B). Both were grown 16+ hours. OD of 0.375 and around 0.650 were deemed OK (still in the growing phase). They were processed and stocked according to the exact protocol from openwetware except that final ODs where 1.6 for -, 0.25 for ?, 0,35 for A, 0.7 for B. protocol wants 1-1.5. |
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| + | Proceeded to do a transfromation with the newly made and frozen(-80°) Top10 cells. Protocol from Invitrogen with 50ul of cells. We did each Top10 batch (-,?,A,B) with PUC19 and X. We also used DH5alpha cells coupled to PHD2, PHD3 and PSND and a control. | ||
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Check plates done yesterday and culture whichever worked, but only if the control worked: | Check plates done yesterday and culture whichever worked, but only if the control worked: | ||
The control showed some colonies. High detection plasmid I (pHD1) also shows some colonies.We recover them and grow them overnight for miniprep and stocking tomorrow. The bacteria transformed from iGEM DNA did not grow. | The control showed some colonies. High detection plasmid I (pHD1) also shows some colonies.We recover them and grow them overnight for miniprep and stocking tomorrow. The bacteria transformed from iGEM DNA did not grow. | ||
Revision as of 15:13, 16 July 2008
Today, Bart is back, we can ask more questions. Also, Alex is back as well.
The cells with the Prey plasmid which have Kan resistance also have a ccdB cassette which is a lethal protein. It is normal that we only recovered Prey cells in CM.
Order of the day :
Prey which had grown turned pink, probably is pseudomonas. We threw it away thinking that, but actually since it was expressing RFP (which our instructor didn't know) it was the normal color. We stocked it in glycerol and did a miniprep to recover the plasmid. We got more than 100 ng/mL of DNA (106 and 168 respectively in the two samples). We also did a stock the pLG cells (100 uL) in glycerol. Miniprep was done of the rest which is not stocked (at least 1mL) to get the plasmid and test the concentration. We g row the already stocked plasmid-transformed cells to do miniprep tomorrow. Try new transformation with some iGEM part which we don't need and adjust protocol. We need to redo the transformation to troubleshoot. Perhaps the protocol which we were using was not the right one, so we modify it according to the iGEM parts registry guidelines (2 hours incubation after the heatshock).
Make competent cells with the Top10 grown before (2 solutions). We divided each solution in two to make to batches(easier for the centrifugation stages). One solution was grown at room temperature on the bench at rest( - and ?). The other was left shaking at 20°C(A and B). Both were grown 16+ hours. OD of 0.375 and around 0.650 were deemed OK (still in the growing phase). They were processed and stocked according to the exact protocol from openwetware except that final ODs where 1.6 for -, 0.25 for ?, 0,35 for A, 0.7 for B. protocol wants 1-1.5.
Proceeded to do a transfromation with the newly made and frozen(-80°) Top10 cells. Protocol from Invitrogen with 50ul of cells. We did each Top10 batch (-,?,A,B) with PUC19 and X. We also used DH5alpha cells coupled to PHD2, PHD3 and PSND and a control.
Check plates done yesterday and culture whichever worked, but only if the control worked:
The control showed some colonies. High detection plasmid I (pHD1) also shows some colonies.We recover them and grow them overnight for miniprep and stocking tomorrow. The bacteria transformed from iGEM DNA did not grow.
