Timeline
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July 20th 2016
- 4 microlitres of milliq water with 1 microlitre of plasmid
- 5 microlitres of no chlorite dismutase
- 1 microlitre of cutsmart buffer (specifically 1.1111 microlitre)
- When adding BsaI, only touch the surface of the BsaI to the pipette tip. The reason: has glycerol which is sticky. Take 0.5 microlitre of BsaI.
- Mix sample very gently then put into a 37 degrees celsius incubator for 1 hour.
- Heat kill at 65 degrees celsius for 20 minutes. This kills the BsaI to prevent star activity.
- Pulse down tubes then add 8 microlitre of milliq water.
- Add 2 microlitres of T4DNA ligase buffer.
- Add 0.5 microlitre of ligase then mix gently and incubate at room temperature for 30 minutes or longer
- a. 10 x cutsmart buffer into tubes makes 1x [ ]
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July 21st 2016
- Did some plating.
- Plated three agar types:
- Purpose: try to find the antibiotic marker on bacteria.
- Observational expectations: If it’s purple, then there is no Cld gene sequence. If white then has CLd gene sequence.
- Lives or dies on certain plates. Alive on ampicillin, then it has the marker. Dies it has the CM marker.
- Added 20 microlitres in each plate
- A. 4 plates of no antibiotic
- B. 10 plates of kanamycin (CM)
- C. 10 plates of ampicillin (Amp)
- A. A total of 7 plates
- B. DNA minus has none, CM, and AMP
- C. Positive control has CM and AMP
- D. Cld +/- has CM and AMP
Competent cell transformation:
- 1 microlitre of positive control added to competent cells (positive control: CPB we used yesterday before cutting and gluing)
- 10 microlitre of cloned plasmid added to competent cells (Cloned plasmid is what we did yesterday).
- Positive control, negative control, and cloned plasmid were chilled for 30 minutes, heat shocked for 90 seconds, chilled on ice for 5 minutes, and finally incubated at 37 degrees celsius with 1 ml of LB broth.
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July 22nd 2016
- It was observed that there were little purple colonies, however the colors will develop more over a longer period.
- Results from yesterday:
- A. No Ab plates: very thick growth
- B. Amp plates: no growth: conclusion: no amp marker
- C. CM: growth
- Results chart:
+ control DNA - Cld + Cld - No Antibiotic Yes, very thick Amp No growth No growth No growth No growth CM Growth (purple colonies) No growth Possibly purple and white* Possible purple and white* -
Mussum ipsum cacilds
Mussum ipsum cacilds, vidis litro abertis. Consetis adipiscings elitis. Pra lá , depois divoltis porris, paradis. Paisis, filhis, espiritis santis. Mé faiz elementum girarzis, nisi eros vermeio, in elementis mé pra quem é amistosis quis leo. Manduma pindureta quium dia nois paga. Sapien in monti palavris qui num significa nadis i pareci latim. Interessantiss quisso pudia ce receita de bolis, mais bolis eu num gostis.
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Mussum ipsum cacilds
Mussum ipsum cacilds, vidis litro abertis. Consetis adipiscings elitis. Pra lá , depois divoltis porris, paradis. Paisis, filhis, espiritis santis. Mé faiz elementum girarzis, nisi eros vermeio, in elementis mé pra quem é amistosis quis leo. Manduma pindureta quium dia nois paga. Sapien in monti palavris qui num significa nadis i pareci latim. Interessantiss quisso pudia ce receita de bolis, mais bolis eu num gostis.
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Mussum ipsum cacilds
Mussum ipsum cacilds, vidis litro abertis. Consetis adipiscings elitis. Pra lá , depois divoltis porris, paradis. Paisis, filhis, espiritis santis. Mé faiz elementum girarzis, nisi eros vermeio, in elementis mé pra quem é amistosis quis leo. Manduma pindureta quium dia nois paga. Sapien in monti palavris qui num significa nadis i pareci latim. Interessantiss quisso pudia ce receita de bolis, mais bolis eu num gostis.
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Mussum ipsum cacilds
Mussum ipsum cacilds, vidis litro abertis. Consetis adipiscings elitis. Pra lá , depois divoltis porris, paradis. Paisis, filhis, espiritis santis. Mé faiz elementum girarzis, nisi eros vermeio, in elementis mé pra quem é amistosis quis leo. Manduma pindureta quium dia nois paga. Sapien in monti palavris qui num significa nadis i pareci latim. Interessantiss quisso pudia ce receita de bolis, mais bolis eu num gostis.