Team:Mexico-UNAM-CINVESTAV/Notebook/Week Five
From 2010.igem.org
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{{Mexico-UNAM-CINVESTAV-HEADER}} | {{Mexico-UNAM-CINVESTAV-HEADER}} | ||
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+ | __NOTOC__ | ||
= Week #5= | = Week #5= | ||
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- | *''' | + | *'''Picked up colonies of transformant cells and set up all for miniprep.''' |
- | ''' | + | ''' We used 30ml de LB medium per falcon tube with 35ng/ml''' |
- | ''' | + | ''' of cloramphenicol and cultured overnight at 37ºC ''' |
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==''Tuesday''== | ==''Tuesday''== | ||
- | |||
- | '''The miniprep was | + | '''The miniprep was prepared using Quiagen Kit.''' |
- | '''Then we | + | '''Then we did a double digestion with ''EcoR''I and ''Pst''I to check out the ligations.''' |
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- | =='' | + | ==''Wednesday''== |
+ | |||
+ | '''We ran a gel to confirm the 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''' | ||
+ | |||
+ | |||
+ | [[Image:Conf3.gif|300px|left]] [[Image:Con1.gif|300px|Right]] | ||
+ | |||
+ | '''We prepared all to repeat the ligation method: PCR2 with Psb1C3 and PCR4 with Psb1C3''' | ||
+ | |||
+ | |||
+ | ==''Thursday''== | ||
+ | |||
+ | |||
+ | '''We did ligations (The LIGator Kit Amresco) PCR2 with Psb1C3 and PCR 4 with Psb1C3, then transformed DH5α''' | ||
+ | |||
+ | '''competent cells, and incubated at 37ºC overnight.''' | ||
+ | |||
+ | '''We ran a gel to test biobricks with the follow results:''' | ||
+ | |||
+ | *'''I0260 Positive Plate 2010 | ||
+ | *'''E0240 Positive Plate 2010 | ||
+ | *'''R010 Positive Plate 2010 | ||
+ | *'''J13002 Negative Plate 2007 | ||
+ | |||
+ | |||
+ | [[Image:Biobricks1.gif]] | ||
+ | |||
+ | |||
+ | ==''Friday''== | ||
+ | |||
+ | '''We checked the previous day ligation plates and we get colonies of''' | ||
+ | |||
+ | '''PCR4 and not of PCR2''' | ||
+ | |||
+ | [[Image:Labo_259.JPG|400px]] | ||
+ | |||
+ | '''Today we discussed the reasons why we have not been succesfull to confirm the PCR2''' | ||
+ | |||
+ | '''maybe the ligation reaction is saturated''' | ||
+ | |||
+ | |||
+ | [[Image:Ligation2.gif]] |
Latest revision as of 22:10, 26 October 2010
Week #5
04th October - 08 October 2010
Monday
- Picked up colonies of transformant cells and set up all for miniprep.
We used 30ml de LB medium per falcon tube with 35ng/ml
of cloramphenicol and cultured overnight at 37ºC
Tuesday
The miniprep was prepared using Quiagen Kit.
Then we did a double digestion with EcoRI and PstI to check out the ligations.
DNA | 5μl |
Buffer NB2 | 1μl |
EcoRI | 0.5μl |
PstI | 0.5μl |
H2O | 2.7μl |
BSA | 0.3μl |
Total | 10μl |
Wednesday
We ran a gel to confirm the 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
![Right](/wiki/images/0/03/Con1.gif)
We prepared all to repeat the ligation method: PCR2 with Psb1C3 and PCR4 with Psb1C3
Thursday
We did ligations (The LIGator Kit Amresco) PCR2 with Psb1C3 and PCR 4 with Psb1C3, then transformed DH5α
competent cells, and incubated at 37ºC overnight.
We ran a gel to test biobricks with the follow results:
- I0260 Positive Plate 2010
- E0240 Positive Plate 2010
- R010 Positive Plate 2010
- J13002 Negative Plate 2007
Friday
We checked the previous day ligation plates and we get colonies of
PCR4 and not of PCR2
Today we discussed the reasons why we have not been succesfull to confirm the PCR2
maybe the ligation reaction is saturated