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Team:Mexico-UNAM-CINVESTAV/Notebook/Week Five
From 2010.igem.org
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*'''Let's picked up colonys our transformant cells and prepare all to do miniprep.''' | *'''Let's picked up colonys our transformant cells and prepare all to do miniprep.''' | ||
| - | '''For this we used 30ml de LB liquid agar medium per falcon tube at | + | '''For this we used 30ml de LB liquid agar medium per falcon tube at 35ng/ml''' |
| - | '''Cloramphenicol concentration | + | '''Cloramphenicol concentration and left overnight culture to 37º ''' |
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==''Tuesday''== | ==''Tuesday''== | ||
| - | '''With a overnight inoculum | + | '''With a overnight inoculum we did a miniprep.''' |
'''The miniprep was done with a Quiagen Kit.''' | '''The miniprep was done with a Quiagen Kit.''' | ||
| - | '''Then we | + | '''Then we did a duble digest with EcoRI and PstI to check out our ligations.''' |
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==''Wensday''== | ==''Wensday''== | ||
| - | '''We | + | '''We ran a gel to confirm our ligations as follow''' |
*'''PCR 1 Lig S with Psb1C3 Confirmed''' | *'''PCR 1 Lig S with Psb1C3 Confirmed''' | ||
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[[Image:Conf3.gif|300px|left]] [[Image:Con1.gif|300px|Right]] | [[Image:Conf3.gif|300px|left]] [[Image:Con1.gif|300px|Right]] | ||
| - | '''We | + | '''We prepared all to repeat ligation; PCR2 with Psb1C3 and PCR4 with Psb1C3''' |
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==''Friday''== | ==''Friday''== | ||
| - | '''We Checked the plates of previous day ligations and we have | + | '''We Checked the plates of previous day ligations and we have cell colonies for''' |
'''PCR4 no for PCR2''' | '''PCR4 no for PCR2''' | ||
Revision as of 18:25, 26 October 2010
Week #5
04th October - 08 October 2010
Monday
- Let's picked up colonys our transformant cells and prepare all to do miniprep.
For this we used 30ml de LB liquid agar medium per falcon tube at 35ng/ml
Cloramphenicol concentration and left overnight culture to 37º
Tuesday
With a overnight inoculum we did a miniprep.
The miniprep was done with a Quiagen Kit.
Then we did a duble digest with EcoRI and PstI to check out our ligations.
| DNA | 5μl |
| Buffer NB2 | 1μl |
| EcoRI | 0.5μl |
| PstI | 0.5μl |
| H2O | 2.7μl |
| BSA | 0.3μl |
| Total | 10μl |
Wensday
We ran a gel to confirm our ligations as follow
- PCR 1 Lig S with Psb1C3 Confirmed
- PCR 2 Lig S with Psb1C3 Unconfirmed
- PCR 3 Lig S with Psb1C3 Confirmed
- PCR 4 Lig C with Psb1C3 Unconfirmed
We prepared all to repeat ligation; PCR2 with Psb1C3 and PCR4 with Psb1C3
Thursday
We did ligations (Fast ligation one hour) Pcr 2 with Psb1C3 and PCR 4 with Psb1C3 then transformed DH5α
competent cells, and incubated at 37º overnight.
We ran a gel to test our biobricks for characterisation with the follow results
- I0260 Positive Plate 2010
- E0240 Positive Plate 2010
- R010 Positive Plate 2010
- J13002 Negative Plate 2007
Friday
We Checked the plates of previous day ligations and we have cell colonies for
PCR4 no for PCR2
Today we discussed the reasons why we have not been succesful to confirm the PCR2
Ligation maybe the reaction is saturated
