Team:UNICAMP-Brazil/Notebooks/October 9

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(→finO and finP with pGEM - confirmation)
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-A PCR: performed with the specific forward primer for our insert and with the reverse primer for pGEM plasmid (M13), in order to confirm that, once our inserts are indeed in there, they are also in the correct frame position.
-A PCR: performed with the specific forward primer for our insert and with the reverse primer for pGEM plasmid (M13), in order to confirm that, once our inserts are indeed in there, they are also in the correct frame position.
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* Seeing that, today we performed the digestion with XbaI and SpeI for all of our minipreps samples that actually worked. Digestion lasted 3 hours.
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* Seeing that, today we performed the digestion with EcoRI and SpeI for all of our minipreps samples that actually worked. Digestion lasted 3 hours.
[[image:Digestao0910_finO_finPempGEM.jpg]]
[[image:Digestao0910_finO_finPempGEM.jpg]]

Revision as of 18:22, 18 October 2009

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finO and finP with pGEM - confirmation

  • We decided that we are going to confirm such ligations by performing:

-A Digestion: with XbaI and SpeI restriction enzymes, in order to release our parts from pGEM plasmid;

-A PCR: performed with the specific forward primer for our insert and with the reverse primer for pGEM plasmid (M13), in order to confirm that, once our inserts are indeed in there, they are also in the correct frame position.

  • Seeing that, today we performed the digestion with EcoRI and SpeI for all of our minipreps samples that actually worked. Digestion lasted 3 hours.

Digestao0910 finO finPempGEM.jpg

  • According to the picture, the digestion excised a fragment of expected size (600 bp for finO and 120 bp for finP) in almost all samples! That's an important clue on confirming our inserts's correctly ligation!


Gabriel and Marcelo




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