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Exeter/4 July 2013
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12. Centrifuge the remaining cells for 2 mins at 13,000 rpm. Pour off the supernatant and resuspend the pellet in 100ul of fresh SOC. Plate this out. | 12. Centrifuge the remaining cells for 2 mins at 13,000 rpm. Pour off the supernatant and resuspend the pellet in 100ul of fresh SOC. Plate this out. | ||
| - | 13. Incubate the plates at 37oC for 12-14 hours overnight | + | 13. Incubate the plates at 37oC for 12-14 hours overnight (make sure they're upside-down!) |
The team divided into 3 groups... | The team divided into 3 groups... | ||
Revision as of 11:53, 15 July 2013
Morning
- Tried to make 1 litre of clear agar using :
5g NaCl
15g Tryptone
15g Agar
5g Soytone
Tryptone, NaCl and soytone were added into a mixer with a magnetic stirrer. (was a yellow colour).
3g of agar were added to two 200ml durans. The Tryptone mix was then added to the durans, filling it up to 200ml.
Mixture was yellow, not colourless - the experiment was not successful. Durans put in the autoclave in the evening to let the agar set overnight.
Afternoon
- Transformation of competent cells
(the standart protocol from iGEM was modified slightly)
1. Competent cells were thawed on ice.
2. 50µl of thawed competent cells was added into a pre chilled 2ml tube, and another 50µl was added to a different 2ml tube which was labelled 'control'.
3. 2µl of the resuspended DNA was added to the 2ml tube. This was pipetted up and down gently a few times. Competent cells were kept on ice.
4. 2µl of the RFP Control was added to the control transformation.
5. Tubes were closed and cells were incubated on ice for 30minutes.
6. The cells were heat shocked by immersion in a pre-heated water bath at 42°C for 60 seconds.
7. Cells were incubated on ice for 5 minutes.
8. SOC media was made by the addition of 500µl of glucose to SOB. 500µl of SOC media was added to each transformation.
9. Cells were incubated at 37°C for 1.5hours while the tubes were shaking.
10. 2 petri dishes were labelled with the part number/control, plasmid backbone and antibiotic resistance.
11. Pipette 100ul of the cells onto one of the plates, and spread using a sterile spread stick.
12. Centrifuge the remaining cells for 2 mins at 13,000 rpm. Pour off the supernatant and resuspend the pellet in 100ul of fresh SOC. Plate this out.
13. Incubate the plates at 37oC for 12-14 hours overnight (make sure they're upside-down!)
The team divided into 3 groups...
- One transformed BBa_I13522, a GFP on pSB1C3
- One transformed BBa_J04450, a RFP on pSBiC3
- One transformed BBa_K322124, which codes for cph8 red light sensor and we need to send off for sequencing.
Overall, each team made
