"
EPF-Lausanne/10 September 2009
From 2009.igem.org
(→Wet Lab) |
(→Wet Lab) |
||
| Line 53: | Line 53: | ||
All have been re-inocculated (500 ul) in 25 ml of fresh M9 + antibiotic, and put in incubator at 37°C at 9.15 am. | All have been re-inocculated (500 ul) in 25 ml of fresh M9 + antibiotic, and put in incubator at 37°C at 9.15 am. | ||
| + | |||
| + | ===Glycerol stocks=== | ||
| + | The 4 "yale" strains have grown (hopefully with no contamination). Note : the strain JW4356 11110 grew in Kana as well -> it's kana resistant. | ||
| + | |||
| + | The other : JW4356 11110 (LB/no antibiotics), JRG465 4456 and JRG1046 5992 all grew in SOB. We will make them competent tonight (100 ml at 3 p.m. at 18°C) and wait until OD = 0.6. | ||
| + | |||
| + | At 2p.m. : the 5 double transformants have been induced at 1mM of IPTG (stock solution : 0.238 g IPTG in 10ml MG). Put at 37°C. | ||
| + | |||
| + | ===Gel=== | ||
| + | 50 ml, 1%, to check the construction BB/Chl (P5B4C5). | ||
| + | |||
| + | '''Result''' : we should see a band at about 1100 bp but we don't. So apparently the vector just religated on itself. We will have to try another colony PCR tomorrow with a positive and a negative control this time ! | ||
==People in the lab== | ==People in the lab== | ||
Revision as of 12:03, 4 October 2009
Contents |
Wet Lab
Colony PCR
We did a colony PCR of the 3 BB / Chl (A, B, C) that have grown (after an overnight + 1 day culture on a plate). It is strange they took so long to grow, so we hope it is not a contamination.
We took a couple of clones on each plate and used the Taq platinium protocol + colony file. We will run a gel to check if the clones are what we wanted.
Results of the culture
All the tubes grew (RO2 / RO1 / double transf.). The medium was only M9 (M9 + AA + thiamine) and not LB this time.
- RO1 #1 (red), #2 (red) which is very nice as it is in M9 (-TRP).
- RO2 #4 (white), #5 (redish), #10 (red). The white one is a good result, but the other should be white as well.
- RO1 + BB #2/3 (white)
- RO1 + BB #3/3 (white)
- RO1 + BB #1/1 (white)
- We have noticed for a few days that RO1#3 doesn't work, there might be a mutation as it never gives the right results.
- The RO1 should be red ! So it could be that the LovTAP is working (upon illumination, LovTAP binds its operator Trp and inhibits TrpO and thus RFP).
- RO2 + BB #8 (RO2 #10 + BB6, clone #8) -> slightly redish
- RO2 + BB #3 (RO2 #4 + BB1, clone #3) -> slightly redish
All have been re-inocculated (500 ul) in 25 ml of fresh M9 + antibiotic, and put in incubator at 37°C at 9.15 am.
Glycerol stocks
The 4 "yale" strains have grown (hopefully with no contamination). Note : the strain JW4356 11110 grew in Kana as well -> it's kana resistant.
The other : JW4356 11110 (LB/no antibiotics), JRG465 4456 and JRG1046 5992 all grew in SOB. We will make them competent tonight (100 ml at 3 p.m. at 18°C) and wait until OD = 0.6.
At 2p.m. : the 5 double transformants have been induced at 1mM of IPTG (stock solution : 0.238 g IPTG in 10ml MG). Put at 37°C.
Gel
50 ml, 1%, to check the construction BB/Chl (P5B4C5).
Result : we should see a band at about 1100 bp but we don't. So apparently the vector just religated on itself. We will have to try another colony PCR tomorrow with a positive and a negative control this time !
People in the lab
Mélanie, Caroline, Basile, Nicolas
