1st experiment in 2007!!

This experiment is related to Rosie's post: "Rachael's boyfriend's plasmid, and Rifampicin"

Back in the year..2005 I have already selected Rif resistant kw20 mutants. As I recall mutant "1" & "i" have only a one point mutation and mutant "R1" & "R2" have two points mutation (confirmed by the Davies' lab) but resist to a higher concentration of Rif than 1 & i.

So here's the plan discussed with Rosie:
Make some overnight culture of 1 or i and R1 or R2 and wildtype kw20 (control) -> take 100ul into 20ml of sBHI -> grow until O.D. = 1.0 -> take 1ml from the flask into a test tube with Map7 DNA -> roll for 15' -> DNase 5' -> series of dilution -> plate on Nov plates sBHI plates.

Result: Compare the TF between RifR mutants & wildtype kw20
When to do this? some time in Jan!

Let me know if I should make change to this plan above. Thanks!!!

cDNA synthesis.. a long LONG day..!

well, I was doing cDNA synthesis (18samples) today.. and that require Reverse Transciptase ($185!!! for only 50ul = ~26samples). So I was adding RT to my samples and it ran out when I still have 4 samples left. I thought "there should be more right?? if not, the other lab should have it..." Then end up neither labs have it and I have to go to class (I was pulling my hair out when no one has RT but Andrew saved the day by helping me again!!! Thanks!!) so I just ran the 14 samples PCR before class (2hrs) and left the rest in the ice bucket until my classes were over. After classes, I went to Wesb to buy the RT and got free chocolate! Which cost way less than the 50ul of RT. So end up I spent more time than I planned :-(

I will be doing cDNA synthesis tomorrow again but I think tomorrow it will run more smoothly than today forsure!!!

I can believe I will be using ~$200 by the end of tomorrow!! I could use that money to buy something else..haha.. but technically that's not my $$$ ! kekeke Anyways, thanks Rosie for the support!~

Pretty Gel!

Today I ran a 1% agarose gel with my 9 samples of RNA that I isolated in the past week or so. It was great! I just can't believe the gel is sooooOoOo nice and pretty! I am just glad all the samples look great. Big thanks to everyone in the lab, especially Andrew who gave me some of his 50X TAE and loading dye to use.




Now that I know my RNA is good, I can go on to cDNA synthesis (next week) and plan out what I am going to do for the next few months. I was scare before that my RNA samples might be bad so I didn't want to promise anything until I know the quality of my RNA.

Time to go back to my homework....

MORE RNA!!!! & lab meeting

Spent another 7ish hrs doing RNA isolation again but this time I did 6 samples instead of 3. Since I have done RNA isolation twice already, I figured out I should be able to handle 6 samples and save some time for other things. Doing 3 samples more took about an extra hour of work but really.. I saved 5 hrs of work if I were to do 3 samples at a time. The bad things about doing 6 samples at a time are my hands get really tired from pipetting (recovering the upper phase step) and I need to be super careful not to mix the samples up. I am just glad is all over... hopefully.. just need to run a gel to check.. if everything look great then today will be my last day of doing RNA isolation!!! (at least in my last undergrad year..hahaha..)

As for lab meeting that just ended: It was great! Wasn't as bad as I thought... What was new about this time was I used diagram to explain my (most of) experiments and that seemed to work pretty well so I will continue to use diagram for my next lab meeting. I hope you all liked the weird food I brought today!

RNA RNA RNA RNA!! (yesterday)

Took me ~6 hours to get some RNA sample... and 2 out of the 3 samples didn't yield as much RNA as I expected. (sample#1: 568.8ng/ul, #2: 976ng/ul & #3: 3421.1ng/ul). So I probably have to concentrate sample #1 & 2 before I can do cDNA synthesis. I should also run a gel next week just to be sure the RNA sample is good...

Just ~1 month before final exams start! Hope to get more done before that... I guess I will stay at the lab during X'mas too. (the undergrad will take over the LAB!! muhahahahahaha )

Lab meeting

After Rosie's lab meeting yesterday.. I am still thinking about why deletion/addition mutation doesn't matter? Is it because eventually after many generations the mutation will cancel each other out (the number of deletion = the number of addition)? or is it sufficient enough to just have substitution mutation alone in the PERL simulation?

Anyhow, I will be presenting next week lab meeting... I don't even remember when was the last time I presented?? Maybe eariler this year?? I still need to think about what results to include and what not.. the presentation will include all the things I have done for the last ~1.5years. I still can't believe it took so long to isolate/find a erythromycin resistant H.influ mutant because it only took about 5 to 6 months to isolate a rifampicin resistant H.influ mutant. So the lab meeting next week will be about what I did to isolate/find this rare eryR mutant!!

What yummylicious food should I bring to lab meeting??? How about some Halloween candy/chocolate? hahaha just kidding.. I will bring something good.. hopefully... :P

Midterms...!

due to mid-terms.. essays... presentation...cold... I was unable to take time to do some exciting experiments. Well.. I can take time to do them but I figured out that if I do my experiments hurryingly, my results might be bad bad bad.. then I have to re-do them again..etc So it is better if I get all my exams and homework done then do some experiments with great results!

Since I don't have experimental data to post.. I'll post something interesting I learned from my neuroscience class!

Botulinum toxin:
A type of botulinum toxin (BOTOX), "A", is used for the beauty treatment for facial lines and wrinkles.

How it works?
The toxin cleave the "snare" proteins needed to have vesicle fusion to occur. So without the fusion, no neurotransimitters are released --> no action potential --> muscles don't move

Good effects?
Your muscles don't move so the wrinkles seem to be gone.

Side effects?
Muscle degeneration! So with continuous use of Botox, your facial muscles will eventually be gone and more Botox will not help with the wrinkles anymore because you don't have any facial muscles left. (paraphrased from my prof)

Conclusion:
BOTOX is bad! We should just be who we are... :-)