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Team:Sevilla/Week1
From 2011.igem.org
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| - | Competent cells:</p> | + | Competent cells:</strong></p> |
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1. Grow cells in tubes with 3 ml of LB o/n at 37ºC with agitation</p> | 1. Grow cells in tubes with 3 ml of LB o/n at 37ºC with agitation</p> | ||
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| - | <p>Transformation</p> | + | <p><strong>Transformation</strong></p> |
<p>1.Let defrost the competent cells in ice.</p> | <p>1.Let defrost the competent cells in ice.</p> | ||
<p>2.Add 2 μL DNA to 50 μL competents cells while working in ice.</p> | <p>2.Add 2 μL DNA to 50 μL competents cells while working in ice.</p> | ||
Latest revision as of 17:01, 21 September 2011
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Monday 11 July
Working in the lab, our first attempt!
After becoming familiar with all the machines, chemicals, pipettes and what seems like thousands of tubes surrounding us, we "tidily" place our own material around the laboratory and set some basic rules for labelling, disposal of waste products and safety stuff. Planning the week is essential, so we spend several hours looking through all the BioBricks we want to use, and working out how we’ll have to mix them to make our bacteria work properly. Restriction enzymes, anti-biotics, promoters, reporters... what wonderful chaos.
Monday Protocols
No protocols used today,but we're gathering some for the next days. Give us some time!
Tuesday 12 July
We prepare the competent cells from the inoculum we made yesterday, following a protocol that seems to be fairly easy and simple. At the same time, some other members of the group shut themselves away in the kitchen to cook up some litres of LB-agar with different antibiotics, and prepare the Petri dishes. Once they're ready, we seed the competent cells.
Tuesday Protocols
Competent cells:
1. Grow cells in tubes with 3 ml of LB o/n at 37ºC with agitation
2. Transfer the cultures to 50 ml of LB and let grow until it reaches an OD of 0,5
3. Cool it in ice for 10 min
4. Transfer 12 ml to steril tubes
5. Centrifuge at 3000rpm for 10 min and remove the supernatant
6. Resuspend cells in CaCl2 100mM (add 5ml to each tube)
7. Add 7ml of CaCl2 100mM (4ºC) to each tube so that the final volume is 12 ml
8. Incubate in ice for 30 min and centrifuge at 4000rpm for 5 min. Remove supernatant.
9. Resuspend all the collected cells in 5 ml of CaCl2: add 5 ml to the first tube, resuspend and put it into the second tube, and so on until all the pellets are contained in the final tube with 5 ml.
10. Store with glycerol at -80ºC (30ul glycerol + 170 ul cell culture)
Wednesday 13 July
To test our competent cells we split into three groups to transform bacteria with all the BioBricks we have, following the enclosed protocol. We seed the transformed bacteria in dishes with the appropriate antibiotic and let them grow overnight. We keep talking about the best way to assemble the BioBricks once we get them. So many things to take into account and so many materials that haven't arrived yet...
Wednesday Protocols
Transformation
1.Let defrost the competent cells in ice.
2.Add 2 μL DNA to 50 μL competents cells while working in ice.
3.Incubate the cells in ice for 30 min
4.Immerse the cells in a bath of 42 ºC for 80 seconds
5.Incubate in ice for 5 min
6.Add 1 mL of LB medium without antibiotics and incubate for 90 min at 37ºC with agitation
7.Centrifuge 5 min at 5000 rpm. Remove 700 μl supernatant and resuspend the pellet.
8.Take two culture dishes with LB agar with the correct antibiotic. Put 50 μl and 200 μl of transformed cells in a dish and make a lawn culture.
9.Incubate 12-14 hours at 37ºC
Thursday 14 July
We entered the laboratory and ran over excitedly to see how our transformations had gone only to discover that none of the bacteria had grown. What a disappointment...We go to the Genetics department to cry a little and get some competent cells so we can start over. They also give us their own protocol to try again, and a control plasmid for transformations (S3139). Competent cells in hand, we do the transformations again with the BioBricks and leave to grow overnight at 37ºC. Hope tomorrow there's something other than LB-agar in those dishes...
Friday 15 July
OH YEAH! There they are! It seems our transformation has been a success. But we don't have all the materials needed to use our new protocol to prepare competent cells, so we spend the evening cooking some more LB, preparing LB-agar dishes and trying to understand how the autoclave works (we are currently enjoying a love-hate relationship with that stupid machine).
Weekend 16-17 July
Sunday evening and it’s 38 ºC in Seville; here we are preparing inoculums of all our transformed cultures to keep them growing in LB+antibiotics o/n at 37 ºC. Who said the weekend was a break?
