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Team:NTU-Singapore/Notebook/3 July 2008
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(New page: Chin Chong & Zhen Fu *Carried out characterization of Standard, Low, Medium and High Promoters that were sent to us in the Newsletter Vol. 1 #Cells that were grown overnight were diluted a...) |
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| + | =Thursday 3 July= | ||
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Chin Chong & Zhen Fu | Chin Chong & Zhen Fu | ||
*Carried out characterization of Standard, Low, Medium and High Promoters that were sent to us in the Newsletter Vol. 1 | *Carried out characterization of Standard, Low, Medium and High Promoters that were sent to us in the Newsletter Vol. 1 | ||
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**Cells fluorescene intensity was directly linked to the strenght of the promoters. The stronger the promoters, the brighter the fluorescene appears to be | **Cells fluorescene intensity was directly linked to the strenght of the promoters. The stronger the promoters, the brighter the fluorescene appears to be | ||
**Pictures and some movie clips were taken | **Pictures and some movie clips were taken | ||
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| + | *E7+Imm successfully PCRed | ||
| + | **Optimizing PCR reaction | ||
| + | ***Due to Self annealing primers(Primers-dimer), Increase Tm value and using 1ul of primers each instead of 2ul | ||
| + | *Staining of gel | ||
| + | **Unsuccessful initially with gel-star stain, however after post soaking of gel in stain buffer for 4hrs, bands became clearer and and missing bands reappeared. | ||
| + | ***New protocol for gel Runs--Post soak of gel in buffer for at least 30 mins | ||
Revision as of 04:23, 4 July 2008
Thursday 3 July
Chin Chong & Zhen Fu
- Carried out characterization of Standard, Low, Medium and High Promoters that were sent to us in the Newsletter Vol. 1
- Cells that were grown overnight were diluted and grown to OD 1.2
- Cells were then centrifuge and the cell pellets were re-suspended with M9 medium with glycerol
- Each cell (promoter) will have two rows of wells assigned to it
- Pipette 200 uL of cells into the respective wells and add 50ul of water to each well
- Take the first sample at time zero and measure subsequent ones at 30 mins intervals using the plate reader at 37 deg C
- Stop the plate reader when extracting the cells from the well, taking note of the RFU value and recording it down on te eppendorf tube containing the extracted cells
- At every 30 mins, take 2 samples from each cell type (the 4 promoters)
- Centrifuge the cells at 4400 rpm, 25 deg C and for 5 mins. Remove the supernantant and store the cell pellets at -20 deg C
- Repeat this for the next 4 hours, collecting 8 sets of data
- One eppendorh tube of each type of cells from the above experiment after the 4 hrs duration, were used as samples to be seen under the fluorecemce microscope
- Cells fluorescene intensity was directly linked to the strenght of the promoters. The stronger the promoters, the brighter the fluorescene appears to be
- Pictures and some movie clips were taken
- E7+Imm successfully PCRed
- Optimizing PCR reaction
- Due to Self annealing primers(Primers-dimer), Increase Tm value and using 1ul of primers each instead of 2ul
- Optimizing PCR reaction
- Staining of gel
- Unsuccessful initially with gel-star stain, however after post soaking of gel in stain buffer for 4hrs, bands became clearer and and missing bands reappeared.
- New protocol for gel Runs--Post soak of gel in buffer for at least 30 mins
- Unsuccessful initially with gel-star stain, however after post soaking of gel in stain buffer for 4hrs, bands became clearer and and missing bands reappeared.
