decent after overnight!

Today's the first day I see decent results.
Conducted microplate reading on Scr and Mism aptamer sequences.

For Mism, the fluorescence intensity decreases as the protein concentration increases. The protein is supposed not to bind. ( not sure. will check)

For Scr, i also dont know.

sleepy. very.

To add to what Ju-nie told me yesterday,
we'll be conducting P2 (or is it microplate reading?) on both scrambled and mismatch DNA aptamer sequence to confirm it's specificity.
Scrambled: Random arrangement of DNA sequences by computer software
Mismatch: Difference only in certain nucleotide bases.
There maybe a consensus sequence between the mismatch and the true DNA which maybe identified through the increment of fluorescent intensity or computer system.

haven't understood the ppt yet. more time needed.

stem loop MB is being used.
Fluoresence intensity to increase in the presence of MRCK.

Orginally they were using FAM and DABCYL as the fluorophore and quencher. (Ex= 480nm, Em= 520nm) Within this wavelength, biological cells are naturally excited hence, not a gd judgement made when measuring fluroesence intensity upon binding of the protein.
Improved to Cy5.5 and BHQ3. Fluoresence intensity increment should be more significant. But, it's the opposite now. It works at wavelength 680nm. A wavelength that cells are not naturally excited.

In microplate reading, sensitivity is set at 150.
Tried changing to S=150 and at Ex= 660nm. not much improvement. (even at higher concentration from 50nM to 500nM)
Increased volume with the addition of more TRIS buffer. (50microlit more). Higher fluoresence intensity observed by the reader.
i dont know the reason still though. now looking at higher volume and higher concentration.

another non intellectual post.
today wasnt the first day i put on my lab coat. but it's the first day i put on the gloves after getting caught.
P2 is still not working. please please work. i want to learn how to do CE!
did dilution using micropipette. learnt about autoclaved pipette tips only are used. dilution factor. manipulating the microplate reader. ( i saw got y-ao sq's folder. wanted to open and see because jc once said she's crossfac so was quite interested to see what she's doing. but i didnt dare to. ) i can only click on 'lfy'

was in the medchem lab for about 2 hours or so. half an hour by myself in there. first time seeing what -83 degree celcius is like. is the north pole that cold? bacteria culture and tissue cultures all about. i decided to wash my jeans even after 1 day. then when this other honours girl opened this thing ( i dont know what it was), my goodness! the whole room stink! was asking what it was. she also dont know.

we didnt get any desired results today! the graphs that came out look like heartbeat scan. and the fluoresence intensity were way too low even after we increased the conc from 50nM to 500nM after 'lunch'. my first day doing something, and also my first day facing such bad results. (in usual labs, anything goes wrong u can get from your friends or from the TA. but here, no friends are doing the exact same thing. something goes wrong, you brainstorm with your mentor or senior) anyway, i contribute to ZERO brainstorming. we'll be increasing the volume tmr.

did dilution for mrck this afternoon. 200nM 400 600 800 1000 2000 4000nM. but still no improvement with increment of conc of MB.

hope tmr will be better. j and j seemed stressed today after the first set of results came out after the first hour. no one seemed interested for lunch, except myself. well, bought lunch for them. i still havent started feeling the stress from undesired results. dont know why.. maybe one day i will. they have succeeded before i came using another molecular structure beacon (just alteration in the fluorophore sequence). unless the quenching this time was that good. or it takes time for the beacon to unfold. let's see the overnight scan tmr. it could have really been the volume because the fluoresence can be even seen by a naked eye. the reader should be able too!

Havent completed more than 1 chapter of CE. Lack of self-discipline.
Was supposed to see more or less how the machine works. Yet, it smoked. Note that it wasn't my fault. Hence, it turned out to be a safety briefing. 2 BIG ring files on safety of lab to be read. Only 1/5 of the File 1 was completed today. To know where the first aid boxes and fire extinguishers are. How to use the fire extinguishers. How to use eye wash. How to use the overhead shower, such that you need to take off eveything. Got to read up on Laser Radiation in addition to CE. Have to complete them before more adds on. Got to learn what the different symbols represent for safety spring check. The only thing I know so far is that skull + 3 crosses = poison. Poison needs lock key. There was also something I should insert my thumb into, or my eardrums were burst.

I'm waiting for all the U-rops students to be finalized for a final safety briefing by Prof S-am Li himself.

More safety readings tommorrow.

1. theory + technique
2. safety
3. theory
4. technique
5. theory.

DNA or RNA aptamers are single-stranded oligonucleotides that can bind proteins, small-molecule compounds and living cells with high affinity and specifity, with matrix conditions optimized. Will it replace the antibodies in the areas of diagnostic, analytical and therapeutic one day?

From the papers I have so far, I can briefly divide the content into 2 broad categories:
1. the method involved in the selection of aptamers (non-SELEX)
2. the behaviour of molecular aptamer beacons upon protein binding

SELEX involves the partitioning of aptamers from non-aptamers by separating target-bound DNA from free DNA and amplification of aptamers by PCR. The technique of non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) will be applied. NECEEM-based non-SELEX for the selection of DNA aptamers rely on electrophoretic separation of DNA-target complexes from free DNA (applicable to targets with relatively high molecular weights, like proteins). We note that in non-SELEX process, no PCR amplification between them is needed. Additional point is non-SELEX provides the opportunity for selection of affinity ligands from DNA-tagged libraries of small molecules and peptides.

In the technique of CE, we assumed that all free DNA molecules have similar electrophoretic mobilities, independent of their sequences. Therefore, only a single electrophoretic zone will be observed for all the free DNA molecules present in the sample. We also assumed that the reattachment of dissociated DNA and Target is negligible due to the high efficiency of separation in NECEEM. SSB may also be included such that SSB will bind to free ssDNA nonspecifically, hence binding nonspecifically free aptamer and precluding the formation of the protein-aptamer complex. (Two kinds of selection: positive and negative) To improve the efficiency, the position and width of the aptamer collection window in CE has to be controlled carefully.

1. Aptamer Design
2. Determination of K_d
3. Determination of the migration time of the apt-prot complex. (take note that separation buffer is used instead of the incubation buffer)
4. Determination of unknown protein concentration (from the migration time)

Advantages of aptamers: Smaller, more stable, can be chemically synthesized. Technique involved here (Fluorescently labelled without affecting their affinity).

Note the difference of DNA library and homogeneous DNA samples.

2kinds of MAB: Quenching and FRET. For each, need to find the binding specificity, binding affinity, detection limit

(Ratiometric imaging)

Incubation conditions to take note of: temp, pH, ionic strength (monovalent or divalent cations have diff effect)

Any enhancement of bioassay?